Inhibition of cancer cell motility

ABSTRACT

Provided herein are compositions and methods for inhibiting cancer cell motility and/or metastasis. In particular embodiments, KBU2046 (or an analog thereof) and one or more additional therapies (e.g., cancer therapies (e.g., hormone therapies and chemotherapies) are provided to inhibit cancer cell motility, inhibit metastasis, and/or treat cancer (e.g., prostate cancer, lung cancer, breast cancer, colon cancer, etc.).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 14/935,040, filed Nov. 6, 2015, which claims priority to U.S.Provisional Patent Application 62/076,297, filed Nov. 6, 2014, which isincorporated by reference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under CA122985 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD

Provided herein are compositions and methods for inhibiting cancer cellmotility and/or metastasis. In particular embodiments, KBU2046 (or ananalog thereof) and one or more additional therapies (e.g., cancertherapies (e.g., hormone therapies, chemotherapies) are provided toinhibit cancer cell motility, inhibit metastasis, and/or treat cancer(e.g., prostate cancer, lung cancer, breast cancer, colon cancer, etc.).

BACKGROUND

The movement of cancer cells out of their primary organ of origingreatly reduces the chances of cure (Wells et al., 2013; hereinincorporated by reference in its entirety). Increased cell motility is aquintessential characteristic of the metastatic phenotype, represents aninitial step in the metastatic cascade, and is absolutely necessary forcancer cells to move from their primary organ of origin to a distantmetastatic site (Talmadge and Fidler, 2010; herein incorporated byreference in its entirety). The development of distant metastases is aprimary cause of the majority of cancer-associated morbidity andmortality (Minn and Massague, 2008; herein incorporated by reference inits entirety). Processes that drive the development of increased cellmotility and metastasis have high potential value as therapeutictargets. However, comprehensive endeavors aimed at selectivelyinhibiting cancer cell motility and resultant metastasis have met withfailure (Coussens et al., 2002; Krishna and Bergan, 2014; Steeg, 2006;herein incorporated by reference in their entireties). While manypathways have been shown to regulate cell motility and metastasis, theyconstitute pathways whose regulatory effects are pleiotropic (Krishnaand Bergan, 2014; herein incorporated by reference in its entirety). Ithas therefore not been possible to identify regulators of cell motilityand metastasis that possess enough selectivity to support their targetedmanipulation.

SUMMARY

Provided herein are compositions and methods for inhibiting cancer cellmotility and/or metastasis. In particular embodiments, KBU2046 (or ananalog thereof) and one or more additional therapies (e.g., cancertherapies (e.g., hormone therapies, chemotherapies) are provided toinhibit cancer cell motility, inhibit metastasis, and/or treat cancer(e.g., prostate cancer, lung cancer, breast cancer, colon cancer, etc.).

Increased cancer cell motility leading to metastasis causes the majorityof cancer-related mortality. Using small molecules as biological probes,we demonstrated that KBU2046, bound within a cleft created byHSP90β/CDC37 heterocomplex formation, stabilized that complex, inhibitedHSP90β-Ser226 phosphorylation, which in turn was shown to inhibit cancercell motility. These molecular perturbations led to inhibition of humancancer cell motility in vitro and of metastasis in murine orthotopicmodels of human prostate and breast cancer metastasis, with effectsobserved at nanomolar concentrations of KBU2046 after oraladministration. Comprehensive molecular, cellular and systemic-basedassays demonstrate that probe action is highly selective for inhibitionof Ser226 phosphorylation, and resultant effects on cell motility andmetastasis.

Experiments conducted during development of embodiments of the presentinvention demonstrated the inhibition of the movement of human breast,prostate, colon and lung cancer cells. Further, proteins that regulatecancer cell movement were identified, and experiments demonstrate thatthey are important pharmacologic targets. Further, specificmodifications to those proteins have been identified that can bepharmacologically inhibited, and thereby inhibit cell movement. Further,experiments demonstrate that therapeutically inhibiting cancer cellmovement can be effectively combined with chemotherapy and hormonetherapy, and that this approach improves the effectiveness of hormonetherapy for human prostate cancer and can be applied to overcome hormoneresistance.

In particular, experiments conducted during development of embodimentsdescribed herein demonstrate that the compound KBU2046: inhibits humanprostate, breast, colon and breast cancer cell motility, inhibits humanprostate and breast cancer metastasis, decreases phosphorylation ofserine 226 on HSP90 β, inhibits cell invasion, and increases binding ofHSP90 to CDC37. Together, these findings demonstrate the HSP90 functioncan be altered through these manipulations. This approach to alteringHSP90 function enhances the efficacy of hormone therapy for treatment ofcancer (e.g., prostate cancer, etc.). This approach to altering HSP90function does not inhibit the efficacy of cytotoxic cancer therapy;rather, data indicates that additive action and their separate actingmechanisms provide systemic synergy of therapies.

Experiments were conducted during development of embodiments describedherein that demonstrate that small molecules can effectively probe thecomplex biology of cancer cell motility and metastasis. Through thisapproach, phosphorylation of Ser²²⁶ on HSP90β has been identified as aregulator of cancer cell motility. Its importance as a pharmacologicallymodifiable regulator of that process has also been demonstrated. Inaddition, a small molecule probe that selectively inhibits Ser²²⁶phosphorylation has been developed, which in turn selectively inhibitscell motility and metastasis. Further, stabilization of the CDC37/HSP90βheterocomplex using a small molecule has been demonstrated. Together,the experiments conducted during development of embodiments describedherein have elucidated a selective and chemically modifiable regulatorymechanism of a critical biological process directly linked to cancermetastasis and its associated high morality.

A significant body of evidence directly supports our proposed model ofKBU2046 binding within a cleft formed by the binding of CDC37 to HSP90β,as depicted in FIGS. 6B, 6C, 6D and FIG. 20. This includes the factsthat KBU2046 does not bind to either CDC37 or HSP90β separately, andonly provides physical stabilization when both CDC37 and HSP90β arepresent. Further, a comprehensive analysis of structural and biophysicalinformation, described in FIGS. 6B, 6C, 6D and FIG. 20, indicates theformation of a cleft in between the two proteins into which KBU2046 canbind without destabilizing stenc interactions. Finally, the function ofKBU2046 is completely distinct from that of classic HSP90 inhibitors(Neckers and Workman, 2012; Whitesell et al., 2012; herein incorporatedby reference in their entireties), consistent with it binding to adistinct site and thereby exerting distinct function. Specifically,classical HSP90 inhibitors induce cellular cytotoxicity (Neckers andWorkman, 2012; Whitesell et al., 2012; herein incorporated by referencein their entireties), whereas this is completely lacking with KBU2046.

The experiments conducted during development of embodiments describedherein demonstrate that phosphorylation of HSP90β Ser226 is an importantregulator of cell invasion, and that compounds or strategies whichmodulate the CDC37-HSP90β interface have important biological andpotential translational relevance. In some embodiments, KBU2046 oranalogues thereof (See, e.g., U.S. Pat. No. 8,481,760 and U.S. Pat. No.8,481,760 8,742,141; herein incorporated by reference in theirentireties) is provided as a cancer treatment, to inhibit cancer cellmotility, to inhibit invasion of pre-cancerous lesions, to inhibitmetastasis, to decrease phosphorylation of HSP90β Ser226, etc. In someembodiments, KBU2046 or analogues thereof are administered to a subjectsuffering from one or more cancers (e.g., solid tumor cancers (e.g.,prostate, lung, breast, colon, etc.)) alone or with one or moreadditional therapies (e.g., hormone therapy, chemotherapy, etc.).

Provided herein are methods for inhibiting cancer cell motility,comprising administering to a subject having cancer a compound havingformula of:

or analogs thereof. In some embodiments, the subject is a human. In someembodiments, the cancer is a solid tumor cancer. In some embodiments,the cancer is selected from the list consisting of prostate, lung,colon, and breast. In some embodiments, the compound is administeredprior to surgical removal of a tumor. In some embodiments, the compoundis administered after surgical removal of a tumor. In some embodiments,the compound is co-administered with a second cancer therapeutic agent.In some embodiments, the second cancer therapeutic agent is a hormonetherapy agent. In some embodiments, the second cancer therapeutic agentis a chemotherapeutic agent. In some embodiments, the administrationinhibits metastasis.

In some embodiments, provided herein are methods for treating a subjectsuffering from cancer and/or inhibiting cancer cell motility, comprisingadministering to a subject having cancer a compound having formula of:

or analogs thereof, to a subject suffering from colon, lung, or breastcancer. In some embodiments, the compound is administered prior tosurgical removal of a tumor. In some embodiments, the compound isadministered after surgical removal of a tumor. In some embodiments, thecompound is co-administered with a second cancer therapeutic agent. Insome embodiments, the second cancer therapeutic agent is a hormonetherapy agent. In some embodiments, the second cancer therapeutic agentis a chemotherapeutic agent. In some embodiments, the administrationinhibits metastasis.

In some embodiments, provided herein are methods of treating a subjectsuffering from cancer and/or inhibiting cancer cell motility, comprisingco-administering: (a) a compound having formula of:

or analogs thereof, and (b) a hormone therapy agent. In someembodiments, the subject suffers from prostate cancer, colon cancer,lung cancer, or breast cancer. In some embodiments, the hormone therapyagent is selected from the list consisting of flutamide, bicalutamide,nilutamide, enzaluatmide, lupron, zoladex, orchiectomy, abiraterone,tamoxifen, raloxifene, anastrozole, fulvestrant, exemestane, letrozole.In some embodiments, (a) and (b) are administered sequentially. In someembodiments, (a) and (b) are administered simultaneously. In someembodiments, (a) and (b) are co-formulated.

In some embodiments, provided herein are compositions comprising aco-formulation of: (a) a compound having formula of:

or analogs thereof, and (b) a hormone therapy agent. In someembodiments, the hormone therapy agent is selected from the listconsisting of flutamide, bicalutamide, nilutamide, enzaluatmide, lupron,zoladex, orchiectomy, abiraterone, tamoxifen, raloxifene, anastrozole,fulvestrant, exemestane, letrozole.

In some embodiments, provided herein are compositions comprising acompound having formula of:

or analogs thereof, linked to a functional moiety via a linker moiety.In some embodiments, compositions comprise a compound having formula of:

wherein L is a linker moiety and R is a functional moiety. In someembodiments, the functional moiety is selected from the list consistingof: an antibody or a fragment thereof, an affinity tag, a peptide orprotein, an oligonucleotide, a solid support, a drug, a metalcoordinating group, a contrast agent, a nanoparticle, a cross-linkinggroup, biotin, a fluorophore, and an immunogenic molecule. Otherexemplary functional moieties for use in the embodiments herein include,but are not limited to: amino acids (e.g., a naturally occurring aminoacid or a non-natural amino acid), a peptide or polypeptide (protein)including an antibody or a fragment thereof, a His-tag, a FLAG tag, aStrep-tag, an enzyme, a cofactor, a coenzyme, a peptide or proteinsubstrate for an enzyme (e.g., branched peptide substrate (e.g.,Z-aminobenzoyl (Abz)-Gly-Pro-Ala-Leu-Ala-4-nitrobenzyl amide (NBA),etc.), a suicide substrate, a receptor, one or more nucleotides (e.g.,ATP, ADP, AMP, GTP or GDP) including analogs thereof, an oligonucleotide(e.g., a double stranded or single stranded DNA), a glycoprotein, apolysaccharide, a peptide-nucleic acid (PNA), a solid support (e.g., asedimental particle such as a magnetic particle, a sepharose orcellulose bead, a membrane, glass (e.g., glass slides), cellulose,alginate, plastic or other synthetically prepared polymer (e.g., aneppendorf tube or a well of a multi-well plate), self-assembledmonolayers, a surface plasmon resonance chip, or a solid support with anelectron conducting surface), a drug (e.g., a chemotherapeutic such asdoxorubicin, 5-fluorouracil, or camptosar (CPT-11; Irinotecan), etc.), apH sensitive agent, a radionuclide, a molecule which is electron opaque,a contrast agent (e.g., barium, iodine or other MM or X-ray contrastagent), a molecule which is sensitive to a reactive oxygen, ananoparticle (e.g., an immunogold particle, paramagnetic nanoparticle,upconverting nanoparticle, or a quantum dot), a nonprotein substrate foran enzyme, an inhibitor of an enzyme, a chelating agent, a cross-linkinggroup (e.g., a succinimidyl ester or aldehyde, glutathione, etc.),biotin or other avidin binding molecule, avidin, streptavidin, cAMP,phosphatidylinositol, heme, a ligand for cAMP, a metal, one or more dyes(e.g., a xanthene dye, a calcium sensitive dye, a sodium sensitive dye,a NO sensitive dye, or other fluorophore), a hapten or an immunogenicmolecule (e.g., one which is bound by antibodies specific for thatmolecule), a radionuclide (e.g., 0.3H, 14C, 35S, 125I, 131I, etc). Insome embodiments, the linker moiety comprises a straight or branchedchain of 1-30 carbon atoms, optionally comprising one or moreheteroatoms and branched or main-chain substituents. In someembodiments, the linker moiety comprises a multiatom straight orbranched chain of atoms selected from C, H, N, O, P, and S. functionalgroups comprising the linker moiety include, but are not limited to—CH₂—, ═CH—, ═C═, CO, CONH, —NH₂, —OH, —SH, —O—, —S—, etc. In someembodiments, the linker moiety comprises one or more (CH₂)₂O groups. Insome embodiments, the linker moiety comprises one or more CONH groups.In some embodiments, the linker moiety comprises (CH₂)₂CONH[(CH₂)₂O]₂ or(CH₂)₄CONH[(CH₂)₂O]₄. In some embodiments, compositions comprise acompound having formula of:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic flow of exemplary probe development strategy.Beginning with 4′,5,7-trihydroxyisoflavone (genistein) as a chemicalscaffold, a fragment-based chemical diversification synthesis approachwas followed, and coupled in an iterative fashion to biological assaysof cell invasion and cell growth inhibition. Compounds that inhibitedcell invasion but did not inhibit cell growth were selected for furthermodification and evaluation. The initial round of synthesis was designedto examine the removal of individual chemical fragments. In this manner,the importance of these functional groups in mediating efficacy(inhibition of cell invasion) was determined. Subsequent rounds builtupon refined structure activity relationship (SAR) knowledge, and soughtto improve efficacy, while deselecting for toxicity (cell growthinhibition). Initial assays were performed with PC3 and PC3-M cells.However, as these studies yielded similar findings, subsequent screeningassays utilized only PC3-M cells. In designing chemical syntheticroutes, priority was given to efficacy and toxicity parameters.Additional design features were also included in our chemical syntheticroutes, but they were only incorporated if they did not compromiseefficacy and toxicity parameters. These additional design featuresincluded removal of fragments that mediated genistein binding to theestrogen receptor (ER), as determined by ER-genistein 3D x-ray crystalstructures (protein Data Base IDs: 1X7R and IX7J, for crystal structuresof ERα and ERβ with bound genistein, respectively). These features alsoincluded removal of chemical fragments that are considered to increasesusceptibility to rapid metabolism, especially that by the cytochromeP450 (CYF) pathway. The final feature involved incorporation of chemicalcharacteristics previously shown to be associated with effective drugsand which together generally impart more favorable pharmacologicproperties, including those described by Lipinski et al. (Lipinski etal., 2001; herein incorporated by reference in its entirety).

FIGS. 2A-E. KBU2046 selectively inhibits cell motility. (FIG. 2A) Cellinvasion. Human prostate metastatic cells (PC3, PC3-M), and HPVtransformed normal (1532NPTX, I542NPTX) and primary cancer (1532CPTX,1542CPTX) cells, were treated with 10 μM genistein (G), KBU2046 (46) orvehicle (CO), and after 3 days, cell invasion was measured. Values aremean±SEM. (FIG. 2B) Single cell migration. Cell migration was measuredafter treatment for 3 days with 10 μM KBU2046 or vehicle (control).Values are mean±SEM. *Denotes Student's t test P value </=0.05, comparedto controls. (FIG. 2C) Cell growth inhibition. The concentrations atwhich KBU2046 or genistein inhibited cell growth after 3 days by 20%(IC20) and 50% (IC50) are depicted, as is the percentage of growthinhibition at 50 μM. Values are mean±SEM. (FIG. 2D) Human cord bloodhematopoietic stem cell colony formation assay. Values are the mean±SDnumber of total, CFU-GM, CFU-GEMM or BFU-E colonies at 14 days aftertreatment with KBU2046, (FIG. 2E) Induction of estrogen responsivegenes. Values are the mean±SD.

FIGS. 3A-D. KBU2046 inhibits cancer metastasis and prolongs life. (A, B)Inhibition of PCa metastasis. Cohorts of athymic mice bearing human PCaPC3-M cell orthotopic implants (A), or of non-tumor bearing athymic mice(B), were treated with KBU2046 incorporated into chow, and resultantlung metastasis (A) or plasma KBU2046 concentration (B) measured. Valuesare the mean±SEM. The relationship between dose and metastasis wasevaluated by two-sided ANOVA (A). (C) Comprehensive characterization ofKBU2046 pharmacokinetics. CD1 mice were dosed with 100 mg KBU2046/kg viaoral gavage or intravenous injection (iv), and blood collected at theindicated time points. The dotted horizontal line denotes aconcentration of 24 nM, which was the concentration of KBU2046 measuredin the blood of mice whose metastasis were suppressed by 92% (A, B). (D)Prolongation of survival in BCa bearing mice. Mice were orthotopicallyimplanted with human breast cancer LM2-4H2N cells, the resultant primarytumors resected, and adjuvant treatment begun with KBU2046 by daily oralgavage five times/week.

FIGS. 4A-E. KBU2046 does not inhibit the MKK4 pathway. (A) Depiction ofestablished MKK4 pathway regulating human PCa cell metastasis. (B)KBU2046 does not bind to MKK4, as measured by fluorescence-based thermalshift assay. Values are the mean±SD increase in melting temperature(ΔTm) of purified recombinant MKK4 induced by the indicatedconcentrations of KBU2046 or genistein. (C) KBU2046 does not inhibitMKK4 in an in vitro kinase assay. The indicated concentrations ofKBU2046 were added to recombinant activated MKK4, and its ability tophosphorylate kinase dead p38a MAPK (K53A) was measured by Western blotfor total (p38 MAPK) and phosphorylated (pp38 MAPK) forms of p38 MAPK.(D, E) KBU2046 does not inhibit downstream phosphorylation of p38 MAPKor of HSP27 in cells. PC3-M cells were pre-treated for 24 hours with 50μM KBU2046 or genistein, as indicated, then with TGFβ, and Western blotperformed.

FIGS. 5A-D. HSP90 β Ser²²⁶ phosphorylation is identified as a regulatorof cell invasion and a mediator of KBU2046 efficacy. (A) Probing forKBU2046-induced changes in protein phosphorylation. PC3-M or PC3 cellswere pre-treated with KBU2046, then with ±TGFβ and the resultant celllysate probed for changes in protein phosphorylation with theKinomeView® assay. The depicted Western blot utilizes KinomeView®phospho-motif antibody, BL4176; the arrow denotes an 83 kDa band whosephosphorylation is inhibited by KBU2046. (B) Proteomic analysis. PC3cells were pre-treated with KBU2046 or vehicle, then with TGFβ, proteinsfrom the resultant cell lysate were immunoprecipitated with BL4176, andHSP90β was identified by LC-MS/MS analysis. The phospho-motif recognizedby the antibody is underlined; S*—denotes Ser226, phosphorylation ofwhich is decreased by KBU2046. (C, D) The phosphorylation status ofHSP90β Ser₂₂₆ regulates human PCa cell invasion and is necessary forKBU2046 action. PC3-M cells were transfected with S226A-, S226D-, orWT-HSP90β, or empty vector (VC), treated with KBU2046 or vehicle, andcell invasion measured. Values are the mean±SEM of a representativeexperiment of multiple experiments.

FIGS. 6A-D. KBU2046 stabilizes CDC37/HSP90β heterocomplexes. (A) KBU2046stabilizes HSP90β/CDC37 heterocomplexes in a DARTS assay. Equimolaramounts of HSP90B and CDC37 protein were pre-incubated with KBU2046, andresultant themlolysin reaction products were detected by silver stainfollowing SDS-PAGE. The mean value of protein bands indicated by arrowsis displayed below each lane, and are expressed as the percentage ofuntreated controL ANOYA P values for changes in band intensity withconcentration are displayed. (B) In-silico model of CDC37 and HSP90βdepicting KBU2046 hydrogen bonding with Gln119 of HSP90β. (C) Lipophilicpotential surface of the computed ligand binding pocket of theCDC37/HSP90β model with KBU2046 bound. (D) Potential surface of thewhole CDC37/HSP90β dimer.

FIG. 7A-U. Exemplary synthesis of KBU2046.

FIGS. 7A-H. Synthetic round #1. As this scaffold had anti-invasionefficacy, it was first evaluated which of its chemical fragments werenecessary for activity by synthesizing a set of compounds lackingindividual functional groups, and assessing their effects upon cellinvasion and cell growth inhibition. Informative findings include: thering C4′-hydroxyl group is necessary for activity (compare compounds 1and 2) and removal of the C7⋅hydroxyl group (which mediates binding tothe ER) does not affect activity (compare compounds 2 and 8). Otherrelevant findings: movement of the C4′-hydroxyl is associated withretention of activity (compare compounds 2 and 5), and it is possible toachieve growth inhibition while having minimal impact upon invasion;consider compounds 1, 3, 4, and 6. Demethylation within the cell cannotbe predicted. Therefore, loss of function was only considered to beinformative for methylated compounds. For example, compound 7, wheremethylation of the C4′⋅hydroxyl group leads to loss of invasion (e.g.,compared to compound 8). This further evidences the importance of theC4′-hydroxyl for activity. In contrast, while the C7- and C4′-hydroxylgroups of compound 16 are methylated, it retains anti-invasion activity,indicating that demethylation within the cell could possibly influencethe results. For cell invasion, PC3-M cells were treated with 10compound for a total of 3 days, and cell invasion assays were run at theend of the 3 day period, in the presence of compound. Values are themean±SEM. Three day MTT cell growth inhibition assays were performedwith PC3-M cells. Values are the mean±SEM.

FIGS. 7J-M. Synthetic round #2. Initial structure-activity relationship(SAR) data informed the second round of compound synthesis. Keybiological findings: it is possible to retain anti-invasion efficacywhile having minimal effect upon cell growth inhibition (compound 22).Additionally, reduction of the C2-C3 double bond does not confer loss ofactivity (compound 22) and appears to reduce off-target cell toxicity.Other findings: moving the C4 carbonyl group to generate the coumarincore confers loss of activity (e.g., compounds 23, 24 and 25).

FIGS. 7N-Q. Synthetic round #3. Substitution of the C4′-hydroxyl groupwith a halide is associated with maintenance of activity (compounds 37and 38). A chemical entity is with potent anti-invasive effects, butwhich still retains growth inhibitory effects is identified (compound38).

FIGS. 7R-U. Synthetic round #4KBU2046 (compound 46), has been identifiedwith anti-invasive efficacy at least equal to that of the startingcompound, 4′,5,7-trihydroxyisoflavone, but that has no growth inhibitoryeffects. Compared to 4′,5,7-trihydroxyisoflavone, KBU2046 is non-planar,lacks hydroxyl groups, and particularly those that mediate ER binding,is halogen-substituted, and has a distinctly different biologicalprofile. These characteristics place KBU2046 in a chemically distinctclass, compared to the starting compound.

FIG. 8. KBU2046 has minimal-to-no cell toxicity in the NCI 60 cell linepanel. KBU2046 was submitted to the Developmental Therapeutics Program(DTP) of the US National Cancer Institute (NCI), underwent initialscreening across the NCI 60 cell line panel per DTP protocol (Shoemaker,2006; herein incorporated by reference in its entirety), and theresultant COMPARE diagram is depicted. Based upon its lack of celltoxicity, NCI did not select KBU2046 to go on to multi-dose testing.

FIG. 9. KBU2046 does not activate the estrogen receptor (ER). Estrogenreceptor positive MCF-7 cells were transfected pERE-Luc or empty controlvector, along with constitutive active β-gal, grown under estrogen-freeconditions, and pre-treated for 24 hours with nanomolar concentrationsof estradiol, or with micromolar concentrations of genistein or KBU2046,as indicated. Luciferase activity was measured, normalized to that ofβ-gal, and values expressed as the percent of untreated vector controlcells. Values are the mean±SD.

FIG. 10. Chemical properties of KBU2046 that favor its ability to reachthe cellular target when delivered systemically. In order for smallcompound probes to exert biological efficacy at the systemic level, theymust be able to reach their protein target inside the body, and thusthey must possess a favorable pharmacologic profile. Recognized chemicalproperties associated with favorable pharmacologic attributes (Lipinskiet al., 2001; herein incorporated by reference in its entirety) areprovided in the table, as are the associated chemical properties ofKBU2046.

FIG. 11. Extensive pharmacokinetic (PK) analysis of KBU2046. CD1 micewere dosed with 25 or 100 mg/kg KBU2046 via oral gavage or intravenousinjection (iv), and blood collected at 0 (pre-dose), 5, 10, 15, 30, 45,60, 90, 120, 180, 240, 360, 480 and 960 minutes post dose. (top graph)Concentration versus time plot, expressed as ng/ml. The 100 mg dosingdata was re-plotted and expressed as nM. (bottom table) The resultantpharmacokinetic parameters. Values are parameter estimates from a naivepooled data approach in which single plasma concentrations measured forindividual animals were pooled for both routes of administration of bothdoses and modeled simultaneously.

FIG. 12. Graph demonstrating that KBU2046 does not inhibit primary tumorcell growth.

FIG. 13. KBU2046 treatment is not associated with systemic off-targeteffects. For histologic examination of tissue, cohorts male 6-8 week oldBalb/c athymic mice (Charles River Laboratories), which did not receiveorthotopic implants, were treated with. After 35 days of treatment, thefollowing organs were harvested at necropsy, and stained with H&E(alternative staining methods as indicated): heart, lungs, esophagus,stomach, colon, small intestine, liver (Trichrome staining), kidneys(Trichrome staining), adrenals, bladder, prostate, spleen, pancreas,brain, testes, and bone marrow and peripheral blood (Giemsa staining).Organs were examined for damage using a semi-quantitative histologicalscoring system (Knodell et al., 1981; herein incorporated by referencein its entirety). No organ damage was observed, except in the livers ofboth control and treatment mice. Changes in the liver observed incontrol mice were not increased by KBU2046 treatment. Mice wereimmunocompromised, and changes in the liver reflected episodic and minorfoci of necrosis, consistent with a prior resolved infection;clinically, mice were all healthy. For examination of organ function,studies used cohorts of 22-24 gm CD1 (ICR) mice (Charles River). Notethat for 22-24 gm/mouse, this translated to 5-7 week old females and4.5-5.5 week old males. Mice were dosed once intravenously with KBU2046at 0 (vehicle), 15, 75 or 125 mg/kg-body weight. On day 8 and 14, allcritical organs were weighed, and the following parameters measured inblood: cholesterol, triglycerides, alanine aminotransferase (ALT),aspartate aminotransferase (AST), total bilirubin, glucose, phosphorus,total protein, calcium, blood urea nitrogen (BUN), creatinine, albumin,Na, K, Cl, white blood cells (with differential), red blood cells,hemoglobin, platelets. No abnormal alterations in any of theseparameters were observed, and there were no significant differencesbetween treatment and control mice.

FIG. 14A-D. Proteomic analysis of the effects of KBU2046 on the kinome.Screening for effects on the kinome. PC3-M cells were pre-treated with10 μM KBU2046, genistein or vehicle control for 3 days, then with ±TGFB,as indicated. Resultant cell lysate, as well as lysate from tumors frommice treated with 150 mg/kg KBU2046 or control mice (from manuscriptFIG. 3A), were then probed with the KinomeView® panel of antibodies byWestern blot. In instances where KBU2046 was inhibiting proteinphosphorylation in cells and in tumors, a repeat experiment of PC3-Mcells was conducted. In addition to including PC3-M cells, experimentswere expanded to examine effects on PC3 cells. (A) The identification ofan 83 kDa band of interest constitutes the only change that wasrepeatable across multiple experiments, and it was observed in PC3 andPC3-M cells, as well as in tumor tissue. (B) Bands of initial interestthat did not repeat. (C,D) All other Western blots of phospho-motifantibodies that were evaluated on initial screen. NI=tumor notinformative; this denotes a tumor sample that yielded an abnormalcoomassie blue staining pattern.

FIG. 15. KBU2046 exhibits greater efficacy under conditions of TGFβtreatment. The invasion of PC3-M cells pretreated with KBU2046 orvehicle control (CO) and then with ±TGFβ was measured.

FIG. 16. Identification of the 83 kDa band using a proteomic approach.PC3 cells were pre-treated with 10 μM KBU2046 or vehicle (control) for 3days, then treated with TGFβ for 1 hr, and the resultant cell lysateswere subjected to immunoprecipitation with BL4176 (Kinoview®phospho-motif antibody). Proteins bound in this manner were analyzedusing PhosphoScan™ technology. This identified 483 unique phosphopeptideassignments from 306 parent proteins, with a mean false discovery rateof 0.30% (estimated via Sorcerer search of composite human database offorward and reverse protein entries). Only proteins where the averagevalues for treatment and control were each 3 times that of backgroundwere considered. Further, it was required that each of the replicatevalues (used to calculate the average) to be >/=2.5 fold abovebackground. According to these parameters, there were 19phospho-proteins whose expression decreased in cells treated withKBU2046, compared to control.

FIG. 17. Knockdown of HSP90β decreases cell invasion and abrogatesKBU2046 efficacy. PC3-M cells were transfected with siRNA to HSP90β(siHSP90β) or non-targeting siRNA (siCO). (Top) The level of HSP90β andHSP90α (HSP90α) transcript levels were measured by qRTIPCR, andexpressed relative to that of GAPDH. (Bottom) Cells were treated withKBU2046 (46) or vehicle control, and cell invasion measured. Values arethe mean±SEM.

FIG. 18. KBU2046 does bind HSP90β or CDC37. Studies used purifiedrecombinant HSP90B or CDC37. There was no evidence of KBU2046 binding toeither HSP90β or CDC37 as measured by isothermal titration calorimetryor by fluorescence-based thermal shift assay. For DARTS assay, HSP90β orCDC37 were individually pre-incubated with KBU2046, thennolysin added,and reaction products were separated by SDS PAGE and visualized bysilver stain (depicted above). NTco=no thennolysin control.

FIGS. 19A-D. KBU2046 binds to intact cells, but not to isolatedproteins. (A) Chemical structure of KBU2046 linked to biotin(KBU2046-biotin). (B) KBU2046-biotin is biologically active. PC3-M cellswere pre-treated with 10 μM KBU2046 or with KBU2046-biotin for threedays, and single cell motility assays conducted. Data are the mean±SEM.(C) KBU2046-biotin labels cells in a manner that can be competed off.PC3-M cells were labeled with 1 KBU2046-biotin+/−10 μM free KBU2046,followed by detection with FITC-streptavidin, and visualization byfluorescent microscopy (with equal exposure times). (D) Protein arrayhybridization. KBU2046-biotin was hybridized to ProtoArray® HumanProtein Microarray's at 0.5 and 10 μM with and without 10-fold excessfree KBU2046. Proteins were first sought that met the followingcriteria, and did so at both 0.5 and 10 μM concentrations ofKBU2046-biotin (in the absence of free KBU2046): Z-Score greater than2.5, Z-Factor greater than 0.5, CI P-value less than 0.05, negativecontrol value <2,000 (relative fluorescence units; RFUs), and asignal/negative control signal of >10 and >5 for 10 μM and 0.5 μMconditions, respectively. This yielded 3 proteins, shown in table, foran initial hit rate of 0.03%. It was then required that free KBU2046inhibit binding by >75% of the two remaining candidates, eliminatingisovaleryl-CoA dehydrogenase (ICD). Its binding signal doubled on goingfrom 0.5 to 10, while percent competition by free KBU2046 markedlydecreased; non-specific binding was suspected by the biotin-linkermoiety. The positive control used in protein arrays was staurosporine.Staurosporine is similar to genistein in that both are small compoundnatural products that are broad spectrum kinase inhibitors. In contrastto the lack of binding by KBU2046-biotin, staurosporine bound to 214proteins at levels that were >/=10 fold above that of background. Thevast majority of these proteins were kinases. Both HSP90 β and CDC37were present on the protein arrays, and were not bound byKBU2046-biotin.

FIG. 20. The HSP90 nucleotide binding site surface (Panel A) shown withbound inhibitor (Wright et al., 2004; herein incorporated by referencein its entirety). When complexed with CDC37, a large cleft is formed atthe interface (Panel B). The CDC37 Arg167 residue dissects the cleftinto two distinct subpockets (Panel C). The nucleotide binding surface(C) is preserved, but a new sub-pocket is formed. KBU2046 is showndocked into the newly formed site (C). The KBU2046 compound was dockedinto the newly formed pocket. A suite of docking software, representingdifferent methodologies and approaches was applied. When allowed in thedocking procedures, side chains from the HSP90 β-CDC37 complex wereallowed to be fully flexible. A consensus pose was reached with rootmean square distance (RMSD) less than 1.1 Angstroms over all atoms thatexhibits no steric clashing with the complex. This model suggests thatthe molecule is capable of binding to this secondary site. A dimer ofthe HSP90β structure in the closed conformation was modeled from S.cerevisiae HSP90A (PDB id=2cg9). In construction of the dimer, the HSP90β-CDC37 interface interactions were maintained. Position and orientationof the extended CDC37 regions were guided by crosslinking data thatshowed inter-domain cross-links between residues 53-347, 107-347, and69-286. This resulting structure shows agreement with other reportedconformations (Vaughan et al., 2006; herein incorporated by reference inits entirety). In this model, both the ATP and proposed KBU2046 pocketsremain intact in the dimerized complex.

FIGS. 21A-C. KBU2046 disrupts osteonectin expression. PC3 cells weretreated+/−with 10 uM KBU2046×3d (A), or were transfected with siRNA toosteonectin (siOST) or non-targeting (siCO) (B), and osteonectin/GAPDHexpression measured by qRT/PCR. (C) PC3 cells were transfected withsiOST, siCO, treated with +/−KBU2046, and cell invasion measured.

FIGS. 22A-C. KBU2046 binds to HSP90β/CDC37 heterocomplexes and inhibitsinvasion by inhibiting HSP9013 Ser²²⁶ phosphorylation. (A) KBU2046stabilizes HSP9013 and CDC37 proteins in a DARTS assay. Thermolysin wasadded to an equimolar mixture of recombinant purified HSP90β and CDC37pre-incubated with KBU2046, followed by silver stain-based detection.The mean value of protein bands, indicated by arrows, is displayed beloweach lane. P values are ANOVA. (B) KBU2046 stabilizes HSP90β/CDC37/CK.HSP90β, CDC37 and casein kinase (CK), were added in an in vitro kinaseassay, followed by Western blot for phospho-serine (P-ser) or with Abspecific for phosphorylated ser226 on HSP90β (P-ser226). The HSP90βprimary degradation product exposes serine residues that CK candifferentially access. In bottom panel, signal is shown to be dependentupon presence of CK. (C) In-silico model of KBU2046 bound to aCDC37-HSP90β heterocomplex.

FIGS. 23A-C. KBU2046 biotin linker. (A) Chemical structure of KBU2046linked to biotin (KBU2046-biotin). (B) KBU2046-biotin is biologicallyactive. PC3-M cells were pre-treated with 10 μM KBU2046 or withKBU2046-biotin for three days, and single cell motility assaysconducted. (C) KBU2046-biotin stains cells and is competed off by10-fold free KBU2046.

FIGS. 24A-D. KBU2046 inhibits the AR-chaperone pathway, andandrogen-driven signaling and growth. (A) KBU2046 decreases binding of a˜62 kDa protein to HSP90β. PC3-M cells were transfected withFLAG-HSP90β, treated for 24 hr with 10 uM KBU2046, FLAG-HSP90β IP'ed,and protein detected by silver stain. (B) KBU2046 decreases binding ofHOP to HSP90β. The same conditions (A), followed by Western blot forHOP. (C) KBU2046 inhibits AR transcriptional activity. LNCaP cells, incharcoal stripped serum (CSS) treated with 10 uM KBU2046 (46), 10 uMbicalutamide (B) or 1.0 nM R1881, and 24 hr later PSA measured byqRT/PCR (normalized to GAPDH and to untreated controls). (D) KBU2046inhibits androgen-driven growth and increases bicalutamide efficacy.LNCaP and VCaP cells were pre-incubated×3 days in CSS, then treated asindicated for either 3 or 6 days, and cell number measured.

DEFINITIONS

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the term “subject suspected of having cancer” refers toa subject that presents one or more symptoms indicative of a cancer(e.g., a noticeable lump or mass) or is being screened for a cancer(e.g., during a routine physical). A subject suspected of having cancermay also have one or more risk factors. A subject suspected of havingcancer has generally not been tested for cancer. However, a “subjectsuspected of having cancer” encompasses an individual who has received apreliminary diagnosis (e.g., a CT scan showing a mass) but for whom aconfirmatory test (e.g., biopsy and/or histology) has not been done orfor whom the stage of cancer is not known. The term further includespeople who once had cancer (e.g., an individual in remission). A“subject suspected of having cancer” is sometimes diagnosed with cancerand is sometimes found to not have cancer.

As used herein, the term “subject diagnosed with a cancer” refers to asubject who has been tested and found to have cancerous cells. Thecancer may be diagnosed using any suitable method, including but notlimited to, biopsy, x-ray, blood test, and the diagnostic methods of thepresent invention. A “preliminary diagnosis” is one based only on visual(e.g., CT scan or the presence of a lump) and/or molecular screeningtests.

As used herein, the term “initial diagnosis” refers to a test result ofinitial cancer diagnosis that reveals the presence or absence ofcancerous cells (e.g., using a biopsy and histology).

As used herein, the term “tumor tissue” refers to a cancerous tissuewithin a subject and may be further designated as “prostate tumortissue,” “lung tumor tissue,” “breast tumor tissue,” etc., according tothe location of origin of the cells within the tumor. In someembodiments, the tumor tissue is “post-surgical tumor tissue,” whichrefers to cancerous tissue that has been removed from a subject (e.g.,during surgery).

As used herein, the term “identifying the risk of said tumormetastasizing” refers to the relative risk (e.g., the percent chance ora relative score) of a tumor metastasizing.

As used herein, the term “identifying the risk of said tumor recurring”refers to the relative risk (e.g., the percent chance or a relativescore) of a tumor recurring in the same organ as the original tumor.

As used herein, the term “subject at risk for cancer” refers to asubject with one or more risk factors for developing a specific cancer.Risk factors include, but are not limited to, gender, age, geneticpredisposition, environmental exposure, and previous incidents ofcancer, preexisting non-cancer diseases, and lifestyle.

As used herein, the term “characterizing cancer in subject” refers tothe identification of one or more properties of a cancer sample in asubject, including but not limited to, the presence of benign,pre-cancerous or cancerous tissue and the stage of the cancer.

As used herein, the term “stage of cancer” refers to a qualitative orquantitative assessment of the level of advancement of a cancer.Criteria used to determine the stage of a cancer include, but are notlimited to, the size of the tumor, whether the tumor has spread to otherparts of the body and where the cancer has spread (e.g., within the sameorgan or region of the body or to another organ). Several stagingmethods are commonly used.

As used herein, the term “characterizing tissue in a subject” refers tothe identification of one or more properties of a tissue sample (e.g.,including but not limited to, the presence of cancerous tissue, thepresence of pre-cancerous tissue that is likely to become cancerous(such as prostatic intraepithelial neoplasia, or PIN), and the presenceof cancerous tissue that is likely to metastasize).

As used herein, the term “providing a prognosis” refers to providinginformation regarding the impact of the presence of cancer (e.g., asdetermined by the diagnostic methods of the present invention) on asubject's future health (e.g., expected morbidity or mortality, thelikelihood of getting cancer, and the risk of metastasis).

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Environmental samplesinclude environmental material such as surface matter, soil, water, andindustrial samples. Such examples are not however to be construed aslimiting the sample types applicable to the present invention.

DETAILED DESCRIPTION

Provided herein are compositions and methods for inhibiting cancer cellmotility and/or metastasis. In particular embodiments, KBU2046 (or ananalog thereof) and one or more additional therapies (e.g., cancertherapies (e.g., hormone therapies) are provided to inhibit cancer cellmotility, inhibit metastasis, and/or treat cancer (e.g., prostatecancer, lung cancer, breast cancer, colon cancer, etc.).

In some embodiments, provided herein are therapeutic compositionscomprising one or more agents for inhibiting cell motility and/ormetastasis (e.g., KBU2046). In some embodiments, methods are provided oftreating cancer, inhibiting cancer cell motility, inhibiting metastasis,prolonging human life etc. through the administration of such compounds(or co-administration).

In addition, agents described herein (e.g., KBU2046, and analogsthereof) may be used together with other therapeutic agents, including,but not limited to, salicylates, steroids, immunosuppressants,chemotherapeutics, hormone therapy, antibodies or antibiotics.Particular therapeutic agents which may be used include, but are notlimited to, the following agents: azobenzene compounds (U.S. Pat. No.4,312,806, incorporated herein by reference), benzyl-substitutedrhodamine derivatives (U.S. Pat. No. 5,216,002, incorporated herein byreference), zinc L-carnosine salts (U.S. Pat. No. 5,238,931,incorporated herein by reference), 3-phenyl-5-carboxypyrazoles andisothiazoles (U.S. Pat. No. 5,294,630, incorporated herein byreference), IL-10 (U.S. Pat. No. 5,368,854, incorporated herein byreference), quinoline leukotriene synthesis inhibitors (U.S. Pat. No.5,391,555, incorporated herein by reference), 2′-halo-2′deoxy adenosine(U.S. Pat. No. 5,506,213, incorporated herein by reference), phenol andbenzamide compounds (U.S. Pat. No. 5,552,439, incorporated herein byreference), tributyrin (U.S. Pat. No. 5,569,680, incorporated herein byreference), certain peptides (U.S. Pat. No. 5,756,449, incorporatedherein by reference), omega-3 polyunsaturated acids (U.S. Pat. No.5,792,795, incorporated herein by reference), VLA-4 blockers (U.S. Pat.No. 5,932,214, incorporated herein by reference), prednisolonemetasulphobenzoate (U.S. Pat. No. 5,834,021, incorporated herein byreference), cytokine restraining agents (U.S. Pat. No. 5,888,969,incorporated herein by reference), p38 inhibitors (Herberich et al(2008) J. Med. Chem 10.1021/jm8005417; Cuenda et al (1995) FEBS Lett.364:229-33; Jackson et al (1998) J. Pharmacol. Exper. Therapeutics284:687-92; Young et at (1997) J Biol Chem 272:12116-21; Goedert et al(1997) EMBO J 16:3563-71; Buo et al (2005) Bioorg. Medicinal Chem. Lett.16:64-8; WO/2007/126871; Xu et al (2008) FEBS Lett 8:1276-82; eachincorporated herein by reference) and nicotine (U.S. Pat. No. 5,889,028,incorporated herein by reference).

Therapeutic agents (e.g., KBU2046) may be used together with agentswhich reduce the viability or proliferative potential of a cell. Agentswhich reduce the viability or proliferative potential of a cell canfunction in a variety of ways including, for example, inhibiting DNAsynthesis, inhibiting cell division, inducing apoptosis, or inducingnon-apoptotic cell killing. Specific examples of cytotoxic andcytostatic agents include but are not limited to, pokeweed antiviralprotein, abrin, ricin, and each of their A chains, doxorubicin,cisplastin, iodine-131, yttrium-90, rhenium-188, bismuth-212, taxol,5-fluorouracil VP-16, bleomycin, methotrexate, vindesine, adriamycin,vincristine, vinblastine, BCNU, mitomycin, paclitaxel, docetaxel,cabazitaxel, mitoxantrone and cyclophosphamide and certain cytokinessuch as TNF-α and TNF-β. Thus, cytotoxic or cytostatic agents caninclude, for example, radionuclides, chemotherapeutic drugs, proteins,and lectins.

Agents which reduce the viability or proliferative potential of cellswhich are responsive to hormones, such as prostate and breast cancer,can function in a variety of ways including, for example, inhibiting theproduction of hormones, including androgens and estrogens, increasingthe metabolism of hormones, by antagonizing the action of hormones, andby removing or altering the function of the targets of hormones,especially the hormone receptors and their associated co-regulators.Specific examples of hormonal agents include but are not limited to,flutamide, bicalutamide, nilutamide, enzaluatmide, lupron, zoladex,orchiectomy, abiraterone, tamoxifen, raloxifene, anastrozole,fulvestrant, exemestane, letrozole and ovariectomy.

“Treating” within the context of the instant invention, means analleviation, in whole or in part, of symptoms associated with a disorderor disease, or slowing, inhibiting or halting of further progression orworsening of those symptoms, or prevention or prophylaxis of the diseaseor disorder in a subject at risk for developing the disease or disorder.Thus, e.g., treating metastatic cancer may include inhibiting orpreventing the metastasis of the cancer, a reduction in the speed and/ornumber of the metastasis, a reduction in tumor volume of themetastasized prostate cancer, a complete or partial remission of themetastasized prostate cancer or any other therapeutic benefit. As usedherein, a “therapeutically effective amount” of a compound of theinvention refers to an amount of the compound that alleviates, in wholeor in part, symptoms associated with a disorder or disease, or slows,inhibits or halts further progression or worsening of those symptoms, orprevents or provides prophylaxis for the disease or disorder in asubject at risk for developing the disease or disorder.

A therapeutically effective amount of a compound as described hereinused in the present invention may vary depending upon the route ofadministration and dosage form. Effective amounts of invention compoundstypically fall in the range of about 0.001 up to 100 mg/kg/day, and moretypically in the range of about 0.05 up to 10 mg/kg/day. Typically, thecompound or compounds used in the instant invention are selected toprovide a formulation that exhibits a high therapeutic index. Thetherapeutic index is the dose ratio between toxic and therapeuticeffects which can be expressed as the ratio between LD₅₀ and ED₅₀. TheLD₅₀ is the dose lethal to 50% of the population and the ED₅₀ is thedose therapeutically effective in 50% of the population. The LD₅₀ andED₅₀ are determined by standard pharmaceutical procedures in animal cellcultures or experimental animals.

Treatment may also include administering the compounds or pharmaceuticalformulations of the present invention in combination with othertherapies. Combinations of the invention may be administeredsimultaneously, separately or sequentially. For example, the compoundsand pharmaceutical formulations of the present invention may beadministered before, during, or after surgical procedure and/orradiation therapy. Alternatively, the compounds of the invention canalso be administered in conjunction with other anticancer agentsdescribed herein. The specific amount of the additional active agentwill depend on the specific agent used, the type of condition beingtreated or managed, the severity and stage of the condition, and theamount(s) of compounds and any optional additional active agentsconcurrently administered to the subject.

In some embodiments of the invention, one or more compounds (e.g.,KBU2046) and an additional active agent (e.g., hormone therapeutic,chemotherapeutic, etc.) are administered to a subject, more typically ahuman, in a sequence and within a time interval such that the compoundcan act together with the other agent to provide an enhanced benefitrelative to the benefits obtained if they were administered otherwise.For example, the additional active agents can be co-administered byco-formulation, administered at the same time or administeredsequentially in any order at different points in time; however, if notadministered at the same time, they should be administered sufficientlyclose in time so as to provide the desired therapeutic or prophylacticeffect. In some embodiments, the compound and the additional activeagents exert their effects at times which overlap. Each additionalactive agent can be administered separately, in any appropriate form andby any suitable route. In other embodiments, the compound isadministered before, concurrently or after administration of theadditional active agents.

In various examples, the compound (e.g., KBU2046) and the additionalactive agent(s) (e.g., hormone therapeutic, chemotherapeutic, etc.) areadministered less than about 1 hour apart, at about 1 hour apart, atabout 1 hour to about 2 hours apart, at about 2 hours to about 3 hoursapart, at about 3 hours to about 4 hours apart, at about 4 hours toabout 5 hours apart, at about 5 hours to about 6 hours apart, at about 6hours to about 7 hours apart, at about 7 hours to about 8 hours apart,at about 8 hours to about 9 hours apart, at about 9 hours to about 10hours apart, at about 10 hours to about 11 hours apart, at about 11hours to about 12 hours apart, no more than 24 hours apart or no morethan 48 hours apart. In some embodiments, therapies are administeredsequentially, where the separation between administrations is one weekor greater (e.g., 1 week, 2 weeks, 1 month, 2 months, 3 months, fourmonths, and ranges therein). For example, five cycles of monthlychemotherapy are administered, followed by treatment with KBU2046 thenext month. In other examples, the compound and the additional activeagents are administered concurrently. In yet other examples, thecompound and the additional active agents are administered concurrentlyby co-formulation.

In other examples, the compound (e.g., KBU2046) and the additionalactive agents (e.g., hormone therapeutic, chemotherapeutic, etc.) areadministered at about 2 to 4 days apart, at about 4 to 6 days apart, atabout 1 week part, at about 1 to 2 weeks apart, or more than 2 weeksapart.

In certain examples, the compound (e.g., KBU2046) and optionally theadditional active agents (e.g., hormone therapeutic, chemotherapeutic,etc.) are cyclically administered to a subject. Cycling therapy involvesthe administration of a first agent for a period of time, followed bythe administration of a second agent and/or third agent for a period oftime and repeating this sequential administration. Cycling therapy canprovide a variety of benefits, e.g., reduce the development ofresistance to one or more of the therapies, avoid or reduce the sideeffects of one or more of the therapies, and/or improve the efficacy ofthe treatment.

In other examples, the compound (e.g., KBU2046) and optionally theadditional active agent (e.g., hormone therapeutic, chemotherapeutic,etc.) are administered in a cycle of less than about 3 weeks, about onceevery two weeks, about once every 10 days or about once every week. Onecycle can comprise the administration of an inventive compound andoptionally the second active agent by infusion over about 90 minutesevery cycle, about 1 hour every cycle, about 45 minutes every cycle,about 30 minutes every cycle or about 15 minutes every cycle. Each cyclecan comprise at least 1 week of rest, at least 2 weeks of rest, at least3 weeks of rest. The number of cycles administered is from about 1 toabout 12 cycles, more typically from about 2 to about 10 cycles, andmore typically from about 2 to about 8 cycles. In some embodiments, thecompound (e.g., KBU2046) is administered daily for an extended period oftime (e.g., 6 months, 1 year, 2 years, 3, years, 4 year, 5 years, rangestherein).

Courses of treatment can be administered concurrently to a subject, forexample, individual doses of the additional active agents areadministered separately yet within a time interval such that the agent(e.g., KBU2046) can work together with the additional active agents(e.g., hormone therapeutic, chemotherapeutic, etc.). For example, onecomponent can be administered once per week in combination with theother components that can be administered once every two weeks or onceevery three weeks. In other words, the dosing regimens are carried outconcurrently even if the therapeutics are not administeredsimultaneously or during the same day.

The active agent(s) (e.g., KBU2046, hormone therapeutic,chemotherapeutic, etc.) can act additively or, more typically,synergistically. In one example, a first agent (e.g. KBU2046) isadministered concurrently with one or more second active agents in thesame pharmaceutical composition. In another example, a first agent (e.g.KBU2046) is administered concurrently with one or more second activeagents in separate pharmaceutical compositions. In still anotherexample, the inventive compound is administered prior to or subsequentto administration of a second active agent. The invention contemplatesadministration of an inventive compound and a second active agent by thesame or different routes of administration, e.g., oral and parenteral.In certain embodiments, a first agent (e.g. KBU2046) is administeredconcurrently with a second active agent that potentially producesadverse side effects including, but not limited to, toxicity, the secondactive agent can advantageously be administered at a dose that fallsbelow the threshold that the adverse side effect is elicited.

In some embodiments, compositions (e.g., comprising KBU2046) areeffective at inhibiting or reversing resistance to certain agents,particularly hormonal agents. This relates to the facts that KBU2046inhibits the function of HSP90β, that HSP90β maintains hormonereceptors, particularly the androgen receptor, in an active state, andthat several resistance mechanisms involve increasing the expression ofthe androgen receptor, or the expression of mutated androgen receptor.In both instances, KBU2046 removes active androgen receptor.

In some embodiments, compositions described herein (e.g., KBU2046) areuseful for preventing the initial development of cancer, andparticularly so in subjects “at risk” for cancer. Its pharmacologicactions would also make it suitable as a cancer prevention agent (e.g.,because it inhibits cell invasion). The invasion of cancer cells throughthe basement membrane of organs they arise from is a requirement for thedefinition of cancer. Thus, KBU2046 inhibits the initial development ofcancer. For example, men with prostatic intraepithelial neoplasia (PIN),have cancer cells present in their prostate glands. Those cells have notyet invaded the basement membrane. Subjects with PIN are at high riskfor developing prostate cancer. Once cells invade the basement membrane,the diagnosis changes from PIN to prostate cancer. Another term used forcancer that has not invaded the basement membrane is in situ cancer, orcarcinoma in situ (CIS) In situ cancer is seen with prostate, breast(ductal or lobular; DCIS, LCIS), lung, and colon. For each organ, thepresence of CIS puts people at high risk for developing invasive cancer.

Provided herein are pharmaceutical compositions and medicaments whichmay be prepared by combining one or more compounds described herein,pharmaceutically acceptable salts thereof, stereoisomers thereof,tautomers thereof, or solvates thereof, with pharmaceutically acceptablecarriers, excipients, binders, diluents or the like to inhibit or treatprimary and/or metastatic prostate cancers. Such compositions can be inthe form of, for example, granules, powders, tablets, capsules, syrup,suppositories, injections, emulsions, elixirs, suspensions or solutions.Compositions can be formulated for various routes of administration, forexample, by oral, parenteral, topical, rectal, nasal, or via implantedreservoir. Parenteral or systemic administration includes, but is notlimited to, subcutaneous, intravenous, intraperitoneal, andintramuscular injections. The following dosage forms are given by way ofexample and should not be construed as limiting.

For oral, buccal, and sublingual administration, powders, suspensions,granules, tablets, pills, capsules, gelcaps, and caplets are acceptableas solid dosage forms. These can be prepared, for example, by mixing oneor more compounds of the instant invention, or pharmaceuticallyacceptable salts or tautomers thereof, with at least one additive suchas a starch or other additive. Suitable additives are sucrose, lactose,cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates,chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins,collagens, casein, albumin, synthetic or semi-synthetic polymers orglycerides. Optionally, oral dosage forms can contain other ingredientsto aid in administration, such as an inactive diluent, or lubricantssuch as magnesium stearate, or preservatives such as paraben or sorbicacid, or antioxidants such as ascorbic acid, tocopherol or cysteine, adisintegrating agent, binders, thickeners, buffers, sweeteners,flavoring agents or perfuming agents. Tablets and pills may be furthertreated with suitable coating materials known in the art.

Liquid dosage forms for oral administration may be in the form ofpharmaceutically acceptable emulsions, syrups, elixirs, suspensions, andsolutions, which may contain an inactive diluent, such as water.Pharmaceutical formulations and medicaments may be prepared as liquidsuspensions or solutions using a sterile liquid, such as, but notlimited to, an oil, water, an alcohol, and combinations of these.Pharmaceutically suitable surfactants, suspending agents, emulsifyingagents, may be added for oral or parenteral administration.

As noted above, suspensions may include oils. Such oils include, but arenot limited to, peanut oil, sesame oil, cottonseed oil, corn oil andolive oil. Suspension preparation may also contain esters of fatty acidssuch as ethyl oleate, isopropyl myristate, fatty acid glycerides andacetylated fatty acid glycerides. Suspension formulations may includealcohols, such as, but not limited to, ethanol, isopropyl alcohol,hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as butnot limited to, poly(ethyleneglycol), petroleum hydrocarbons such asmineral oil and petrolatum; and water may also be used in suspensionformulations.

Injectable dosage forms generally include aqueous suspensions or oilsuspensions which may be prepared using a suitable dispersant or wettingagent and a suspending agent. Injectable forms may be in solution phaseor in the form of a suspension, which is prepared with a solvent ordiluent. Acceptable solvents or vehicles include sterilized water,Ringer's solution, or an isotonic aqueous saline solution.Alternatively, sterile oils may be employed as solvents or suspendingagents. Typically, the oil or fatty acid is non-volatile, includingnatural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.

For injection, the pharmaceutical formulation and/or medicament may be apowder suitable for reconstitution with an appropriate solution asdescribed above. Examples of these include, but are not limited to,freeze dried, rotary dried or spray dried powders, amorphous powders,granules, precipitates, or particulates. For injection, the formulationsmay optionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these.

For rectal administration, the pharmaceutical formulations andmedicaments may be in the form of a suppository, an ointment, an enema,a tablet or a cream for release of compound in the intestines, sigmoidflexure and/or rectum. Rectal suppositories are prepared by mixing oneor more compounds of the instant invention, or pharmaceuticallyacceptable salts or tautomers of the compound, with acceptable vehicles,for example, cocoa butter or polyethylene glycol, which is present in asolid phase at normal storing temperatures, and present in a liquidphase at those temperatures suitable to release a drug inside the body,such as in the rectum. Oils may also be employed in the preparation offormulations of the soft gelatin type and suppositories. Water, saline,aqueous dextrose and related sugar solutions, and glycerols may beemployed in the preparation of suspension formulations which may alsocontain suspending agents such as pectins, carbomers, methyl cellulose,hydroxypropyl cellulose or carboxymethyl cellulose, as well as buffersand preservatives.

Compounds of the invention may be administered to the lungs byinhalation through the nose or mouth. Suitable pharmaceuticalformulations for inhalation include solutions, sprays, dry powders, oraerosols containing any appropriate solvents and optionally othercompounds such as, but not limited to, stabilizers, antimicrobialagents, antioxidants, pH modifiers, surfactants, bioavailabilitymodifiers and combinations of these. Formulations for inhalationadministration contain as excipients, for example, lactose,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate. Aqueousand nonaqueous aerosols are typically used for delivery of inventivecompounds by inhalation.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the compound together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (TWEENs, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions. A nonaqueous suspension (e.g., in a fluorocarbonpropellant) can also be used to deliver compounds of the invention.

Aerosols containing compounds for use according to the present inventionare conveniently delivered using an inhaler, atomizer, pressurized packor a nebulizer and a suitable propellant, e.g., without limitation,pressurized dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, nitrogen, air, or carbon dioxide. In the caseof a pressurized aerosol, the dosage unit may be controlled by providinga valve to deliver a metered amount. Capsules and cartridges of, forexample, gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch. Delivery of aerosols of the present inventionusing sonic nebulizers is advantageous because nebulizers minimizeexposure of the agent to shear, which can result in degradation of thecompound.

For nasal administration, the pharmaceutical formulations andmedicaments may be a spray, nasal drops or aerosol containing anappropriate solvent(s) and optionally other compounds such as, but notlimited to, stabilizers, antimicrobial agents, antioxidants, pHmodifiers, surfactants, bioavailability modifiers and combinations ofthese. For administration in the form of nasal drops, the compounds maybe formulated in oily solutions or as a gel. For administration of nasalaerosol, any suitable propellant may be used including compressed air,nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent.

Dosage forms for the topical (including buccal and sublingual) ortransdermal administration of compounds of the invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,and patches. The active component may be mixed under sterile conditionswith a pharmaceutically-acceptable carrier or excipient, and with anypreservatives, or buffers, which may be required. Powders and sprays canbe prepared, for example, with excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates and polyamide powder, ormixtures of these substances. The ointments, pastes, creams and gels mayalso contain excipients such as animal and vegetable fats, oils, waxes,paraffins, starch, tragacanth, cellulose derivatives, polyethyleneglycols, silicones, bentonites, silicic acid, talc and zinc oxide, ormixtures thereof.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the invention to the body. Such dosage formscan be made by dissolving or dispersing the agent in the proper medium.Absorption enhancers can also be used to increase the flux of theinventive compound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe compound in a polymer matrix or gel.

Besides those representative dosage forms described above,pharmaceutically acceptable excipients and carriers are generally knownto those skilled in the art and are thus included in the instantinvention. Such excipients and carriers are described, for example, in“Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991),which is incorporated herein by reference.

The formulations of the invention may be designed to be short-acting,fast-releasing, long-acting, and sustained-releasing as described below.Thus, the pharmaceutical formulations may also be formulated forcontrolled release or for slow release.

The instant compositions may also comprise, for example, micelles orliposomes, nanoformulation, or some other encapsulated form, or may beadministered in an extended release form to provide a prolonged storageand/or delivery effect. Therefore, the pharmaceutical formulations andmedicaments may be compressed into pellets or cylinders and implantedintramuscularly or subcutaneously as depot injections or as implantssuch as stents. Such implants may employ known inert materials such assilicones and biodegradable polymers.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions, sex, and diet of thesubject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant invention.

In some embodiments, one or more agents (e.g., KBU2046) are administered(e.g., for inhibiting cell motility, for inhibiting invasion ofpre-cancerous lesions, for inhibiting metastasis, etc.) in conjunctionwith one or more additional treatments, therapies, therapeutics, and/orprocedures (e.g., for treatment of cancer, etc.). Although embodimentsdescribed herein are not limited by the scope of these additionaltreatments, therapies, therapeutics, and/or procedures, they mayinclude, hormone therapy, chemotherapy, radiation, surgery, etc.,suitable agents for use with the agents described herein (e.g. KBU2046and analogues thereof) include the following.

Alkylating agents are chemotherapy agents that attack the negativelycharged sites on the DNA (e.g., the oxygen, nitrogen, phosphorous andsulfur atoms) and bind to the DNA thus altering replication,transcription and even base pairing. It is also believed that alkylationof the DNA also leads to DNA strand breaks and DNA strand cross-linking.By altering DNA in this manner, cellular activity is effectively stoppedand the cancer cell will die. Common alkylating agents include, withoutlimitation, procarbazine, ifosphamide, cyclophosphamide, melphalan,chlorambucil, decarbazine, busulfan, thiotepa, and the like. Alkylatingagents such as those mentioned above can be used in combination with oneor more other alkylating agents and/or with one or more chemotherapyagents of a different class(es).

Platinum chemotherapy agents are believed to inhibit DNA synthesis,transcription and function by cross-linking DNA subunits. (Thecross-linking can happen either between two strands or within one strandof DNA.) Common platinum chemotherapy agents include, withoutlimitation, cisplatin, carboplatin, oxaliplatin, Eloxatin, and the like.Platinum chemotherapy agents such as those mentioned above can be usedin combination with one or more other platinums and/or with one or morechemotherapy agents of a different class(es).

Anti-metabolite chemotherapy agents are believed to interfere withnormal metabolic pathways, including those necessary for making new DNA.Common anti-metabolites include, without limitation, Methotrexate,5-fluorouracil (e.g., capecitabine), gemcitabine(2′-deoxy-2′,2′-difluorocytidine monohydrochloride (β-isomer), EliLilly), 6-mercaptopurine, 6-thioguanine, fludarabine, cladribine,cytarabine, tegafur, raltitrexed, cytosine arabinoside, and the like.Gallium nitrate is another anti-metabolite that inhibits ribonucleotidesreductase. Anti-metabolites such as those mentioned above can be used incombination with one or more other anti-metabolites and/or with one ormore chemotherapy agents of a different class(es).

Anthracyclines promote the formation of free oxygen radicals. Theseradicals result in DNA strand breaks and subsequent inhibition of DNAsynthesis and function. Anthracyclines are also inhibit the enzymetopoisomerase by forming a complex with the enzyme and DNA. Commonanthracyclines include, without limitation, daunorubicin, doxorubicin,idarubicin, epirubicin, mitoxantrone, adriamycin, bleomycin,mitomycin-C, dactinomycin, mithramycin and the like. Anthracyclines suchas those mentioned above can be used in combination with one or moreother anthracyclines and/or with one or more chemotherapy agents of adifferent class(es).

Taxanes bind with high affinity to the microtubules during the M phaseof the cell cycle and inhibit their normal function. Common taxanesinclude, without limitation, paclitaxel, docetaxel, Taxotere, Taxol,taxasm, 7-epipaclitaxel, t-acetyl paclitaxel, 10-desacetyl-paclitaxel,10-desacetyl-7-epipaclitaxel, 7-xylosylpaclitaxel,10-desacetyl-7-epipaclitaxel, 7-N—N-dimethylglycylpaclitaxel,7-L-alanylpaclitaxel and the like. Taxanes such as those mentioned abovecan be used in combination with one or more other taxanes and/or withone or more chemotherapy agents of a different class(es).

Camptothecins complex with topoisomerase and DNA resulting in theinhibition and function of this enzyme. Common camptothecins include,without limitation, irinotecan, topotecan, etoposide, vinca alkaloids(e.g., vincristine, vinblastine or vinorelbine), amsacrine, teniposideand the like. Camptothecins such as those mentioned above can be used incombination with one or more other camptothecins and/or with one or morechemotherapy agents of a different class(es).

Nitrosoureas inhibit changes necessary for DNA repair. Commonnitrosoureas include, without limitation, carmustine (BCNU), lomustine(CCNU), semustine and the like. Nitrosoureas such as those mentionedabove can be used in combination with one or more other nitrosoureasand/or with one or more chemotherapy agents of a different class(es).

EGFR (i.e., epidermal growth factor receptor) inhibitors inhibit EGFRand interfere with cellular responses including cell proliferation anddifferentiation. EGFR inhibitors include molecules that inhibit thefunction or production of one or more EGFRs. They include small moleculeinhibitors of EGFRs, antibodies to EGFRs, antisense oligomers, RNAiinhibitors and other oligomers that reduce the expression of EGFRs.Common EGFR inhibitors include, without limitation, gefitinib, erlotinib(Tarceva), cetuximab (Erbitux), panitumumab (Vectibix, Amgen) lapatinib(GlaxoSmithKline), CI1033 or PD183805 or canternib(6-acrylamide-N-(3-chloro-4-fluororphenyl)-7-(3-morpholinopropo-xy)quinazolin-4-amine,Pfizer), and the like. Other inhibitors include PKI-166(4-[(1R)-1-phenylethylamino]-6-(4-hydroxyphenyl)-7H-pyrrolo[2,3-d-]pyrimidine,Novartis), CL-387785(N-[4-(3-bromoanilino)quinazolin-6-yl]but-2-ynamide), EKB-569(4-(3-chloro-4-fluororanilino)-3-cyano-6-(4-dimethylaminobut2(E)-enamido)-7-ethoxyquinoline,Wyeth), lapatinib (GW2016, GlaxoSmithKline), EKB509 (Wyeth), panitumumab(ABX-EGF, Abgenix), matuzumab (EMD 72000, Merck), and the monoclonalantibody RH3 (New York Medical). EGFR inhibitors such as those mentionedabove can be used in combination with one or more other EGFR inhibitorsand/or with one or more chemotherapy agents of a different class(es).

Antibiotics promote the formation of free oxygen radicals that result inDNA breaks leading to cancer cell death. Common antibiotics include,without limitation, bleomycin and rapamycin and the like. The macrolidefungicide rapamycin (also called RAP, rapamune and sirolimus) bindsintracellularly to the to the immunophilin FK506 binding protein 12(FKBP12) and the resultant complex inhibits the serine protein kinaseactivity of mammalian target of rapamycin (mTOR). Rapamycin macrolidesinclude naturally occurring forms of rapamycin as well as rapamycinanalogs and derivatives that target and inhibit mTOR. Other rapamycinmacrolides include, without limitation, temsirolimus (CCI-779, Wyeth)),everolimus and ABT-578. Antibiotics such as those mentioned above can beused in combination with one or more other antibiotics and/or with oneor more chemotherapy agents of a different class(es).

HER2/neu Inhibitors block the HER2 receptor and prevent the cascade ofreactions necessary for tumor survival. Her2 inhibitors includemolecules that inhibit the function or production of Her2. They includesmall molecule inhibitors of Her2, antibodies to Her2, antisenseoligomers, RNAi inhibitors and other oligomers that reduce theexpression of tyrosine kinases. Common HER2/neu inhibitors include,without limitation, trastuzumab (Herceptin, Genentech) and the like.Other Her2/neu inhibitors include bispecific antibodies MDX-210(FC.gamma.R1-Her2/neu) and MDX-447 (Medarex), pertuzumab (rhuMAb 2C4,Genentech), HER2/neu inhibitors such as those mentioned above can beused in combination with one or more other HER2/neu inhibitors and/orwith one or more chemotherapy agents of a different class(es).

Angiogenesis inhibitors inhibit vascular endothelial growth factor, i.e.VEGF, thereby inhibiting the formation of new blood vessels necessaryfor tumor life. VEGF inhibitors include molecules that inhibit thefunction or production of one or more VEGFs. They include small moleculeinhibitors of VEGF, antibodies to VEGF, antisense oligomers, RNAiinhibitors and other oligomers that reduce the expression of tyrosinekinases. Common angiogenesis inhibitors include, without limitation,bevacizumab (Avastin, Genentech). Other angiogenesis inhibitors include,without limitation, ZD6474 (AstraZeneca), BAY-43-9006, sorafenib(Nexavar, Bayer), semaxanib (SU5416, Pharmacia), SU6668 (Pharmacia),ZD4190(N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[2-(1H-1,2,3-triazol-1-yl)ethoxy]-quinazolin-4-amine,Astra Zeneca), Zactima (ZD6474,N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[2-(1H-1,2,3-triazol-1-yl)ethoxy]quinazolin-4-amine,Astra Zeneca), vatalanib, (PTK787, Novartis), the monoclonal antibodyIMC-1C11 (Imclone) and the like. Angiogenesis inhibitors such as thosementioned above can be used in combination with one or more otherangiogenesis inhibitors and/or with one or more chemotherapy agents of adifferent class(es).

In addition to EGFR, HER2 and VEGF inhibitors, other kinase inhibitorsare used as chemotherapeutic agents. Aurora kinase inhibitors include,without limitation, compounds such as 4-(4-Nbenzoylamino)aniline)-6-methyoxy-7-(3-(1-morpholino)propoxy)quinazoline(ZM447439, Ditchfield et al., J. Cell. Biol., 161:267-80 (2003)) andhesperadin (Haaf et al., J. Cell Biol., 161: 281-94 (2003)). Othercompounds suitable for use as Aurora kinase inhibitors are described inVankayalapati H, et al., Mol. Cancer. Ther. 2:283-9 (2003). SRC/Ablkinase inhibitors include without limitation, AZD0530(4-(6-chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-1-ypethox-y]-5-tetrahycropyran-4-yloxyquinazoline).Tyrosine kinase inhibitors include molecules that inhibit the functionor production of one or more tyrosine kinases. They include smallmolecule inhibitors of tyrosine kinases, antibodies to tyrosine kinasesand antisense oligomers, RNAi inhibitors and other oligomers that reducethe expression of tyrosine kinases. CEP-701 and CEP-751 (Cephalon) actas tyrosine kinase inhibitors. Imatinib mesylate is a tyrosine kinaseinhibitor that inhibits bcr-abl by binding to the ATP binding site ofbcr-abl and competitively inhibiting the enzyme activity of the protein.Although imatinib is quite selective for bcr-abl, it does also inhibitother targets such as c-kit and PDGF-R. FLT-3 inhibitors include,without limitation, tandutinib (MLN518, Millenium), sutent (SU11248,5-[5-fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-py-rrole-3-carboxylicacid [2-diethylaminoethyl]amide, Pfizer), midostaurin (4′-N-benzoylstaurosporine, Novartis), lefunomide (SU101) and the like. MEKinhibitors include, without limitation,2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzami-de)(PD184352/CI-1044, Pfizer), PD198306 (Pfizer), PD98059(2′-amino-3′-methoxyflavone), U0126 (Promega), Ro092-210 from fermentedmicrobial extracts (Roche), the resorcyclic acid lactone, L783277, alsoisolated from microbial extracts (Merck) and the like. Tyrosine kinaseinhibitors such as those mentioned above can be used in combination withone or more other tyrosine kinase inhibitors and/or with one or morechemotherapy agents of a different class(es).

Proteaosome inhibitors inhibit the breakdown of some of these proteinsthat have been marked for destruction. This results in growth arrest ordeath of the cell. Common proteaosome inhibitors include, withoutlimitation, bortezomib, ortezomib and the like. Proteaosome inhibitorssuch as those mentioned above can be used in combination with one ormore other proteaosome inhibitors and/or with one or more chemotherapyagents of a different class(es).

Immunotherapies are thought to bind to and block specific targets,thereby disrupting the chain of events needed for tumor cellproliferation. Common immunotherapies include, without limitation,rituximab and other antibodies directed against CD20, Campath-1H andother antibodies directed against CD-50, epratuzmab and other antibodiesdirected against CD-22, galiximab and other antibodies directed againstCD-80, apolizumab HU1D10 and other antibodies directed against HLA-DR,and the like. Radioisotopes can be conjugated to the antibody, resultingin radioimmunotherapy. Two such anti-CD20 products are tositumomab(Bexxar) and ibritumomab (Zevalin) Immunotherapies such as thosementioned above can be used in combination with one or more otherimmunotherapies and/or with one or more chemotherapy agents of adifferent class(es).

Hormone therapies block cellular receptors, inhibit the in vivoproduction of hormones, and/or eliminate or modify hormone receptors oncells, all with the end result of slowing or stopping tumorproliferation. Common hormone therapies include, without limitation,antiestrogens (e.g., tamoxifen, toremifene, fulvestrant, raloxifene,droloxifene, idoxifene and the like), progestogens) e.g., megestrolacetate and the like) aromatase inhibitors (e.g., anastrozole,letrozole, exemestane, vorozole, exemestane, fadrozole,aminoglutethimide, exemestane, 1-methyl-1,4-androstadiene-3,17-dione andthe like), anti-androgens (e.g., bicalutimide, nilutamide, flutamide,cyproterone acetate, and the like), luteinizing hormone releasinghormone agonist (LHRH Agonist) (e.g., goserelin, leuprolide, buserelinand the like); 5-.alpha.-reductase inhibitors such as finasteride, andthe like. Hormone therapies such as those mentioned above can be used incombination with one or more other hormone therapies and/or with one ormore chemotherapy agents of a different class(es).

Photodynamic therapies expose a photosensitizing drug to specificwavelengths of light to kill cancer cells. Common photodynamic therapiesinclude, for example, porfimer sodium (e.g., Photofrine) and the like.Photodynamic therapies such as those mentioned above can be used incombination with one or more other photodynamic therapies and/or withone or more chemotherapy agents of a different class(es).

Cancer vaccines are thought to utilize whole, inactivated tumor cells,whole proteins, peptide fragments, viral vectors and the like togenerate an immune response that targets cancer cells. Common cancervaccines include, without limitation, modified tumor cells, peptidevaccine, dendritic vaccines, viral vector vaccines, heat shock proteinvaccines and the like.

Histone deacetylase inhibitors are able to modulate transcriptionalactivity and consequently, can block angiogenesis and cell cycling, andpromote apoptosis and differentiation. Histone deacetylase inhibitorsinclude, without limitation, SAHA (suberoylanilide hydroxamic acid),depsipeptide (FK288) and analogs, Pivanex (Titan), CI994 (Pfizer), MS275PXD101 (CuraGen, TopoTarget) MGCD0103 (MethylGene), LBH589, NVP-LAQ824(Novartis) and the like and have been used as chemotherapy agents.Histone deacetylase inhibitors such as those mentioned above can be usedin combination with one or more other histone deacetylase inhibitorsand/or with one or more chemotherapy agents of a different class(es).

Modulators of Sphingolipid metabolism have been shown to induceapoptosis. For reviews see N. S. Raclin, Biochem J, 371:243-56 (2003);D. E. Modrak, et al., Mol. Cancer. Ther, 5:200-208 (2006), K. Desai, etal., Biochim Biophys Acta, 1585:188-92 (2002) and C. P. Reynolds, et al.and Cancer Lett, 206, 169-80 (2004), all of which are incorporatedherein by reference. Modulators and inhibitors of various enzymesinvolved in sphingolipid metabolism can be used as chemotherapeuticagents. Ceramide has been shown to induce apoptosis. Other analogsinclude, without limitation, Cer 1-glucuronide, poly(ethyleneglycol)-derivatized ceramides and pegylated ceramides. Modulators thatstimulate ceramide synthesis have been used to increase ceramide levels.Compounds that stimulate serine palmitoyltransferase, an enzyme involvedin ceramide synthesis, include, without limitation, tetrahydrocannabinol(THC) and synthetic analogs and anandamide, a naturally occurringmammalian cannabinoid. Gemcitabine, retinoic acid and a derivative,fenretinide [N-(4-hydroxyphenyl)retinamide, (4-HPR)], camptothecin,homocamptothecin, etoposide, paclitaxel, daunorubicin and fludarabinehave also been shown to increase ceramide levels. In addition, valspodar(PSC833, Novartis), a non-immunosuppressive non-ephrotoxic analog ofcyclosporin and an inhibitor of p-glycoprotein, increases ceramidelevels. Modulators of sphingomyelinases can increase ceramide levels.They include compounds that lower GSH levels, as GSH inhibitssphingomyelinases. For example, betathine (β-alanyl cysteaminedisulphide), oxidizes GSH, and has produced good effects in patientswith myeloma, melanoma and breast cancer. COX-2 inhibitors, such ascelecoxib, ketoconazole, an antifungal agent, doxorubicin, mitoxantrone,D609 (tricyclodecan-9-yl-xanthogenate), dexamethasone, and Ara-C(1-β-D-arabinofuranosylcytosine) also stimulate sphingomyelinases.Molecules that stimulate the hydrolysis of glucosylceramide also raiseceramide levels. The enzyme, GlcCer glucosidase, which is available foruse in Gaucher's disease, particularly with retinol or pentanol asglucose acceptors and/or an activator of the enzyme can be used astherapeutic agents. Saposin C and analogs thereof, as well as analogs ofthe anti-psychotic drug, chloropromazine, may also be useful. Inhibitorsof glucosylceramide synthesis include, without limitation, PDMP(N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenylethyldecanamide]), PMPP(D,L-threo-(1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol), P4 orPPPP (D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol),ethylenedioxy-P4,2-decanoylamine-3-morpholinoprophenone, tamixofen,raloxifene, mifepristone (RU486), N-butyl deoxynojirimycin andanti-androgen chemotherapy (bicalutamide+leuprolide acetate). Zavesca,(1,5-(butylimino)-1,5-dideoxy-D-glucitol) usually used to treatGaucher's disease, is another inhibitor of glucosylceramide synthesis.Inhibitors of ceramidase include, without limitation,N-oleoylethanolamine, a truncated form of ceramide, D-MAPP(D-erythro-2-tetradecanoylamino-1-phenyl-1-propanol) and the relatedinhibitor B13 (p-nitro-D-MAPP). Inhibitors of sphingosine kinase alsoresult in increased levels of ceramide. Inhibitors include, withoutlimitation, safingol (L-threo-dihydrosphingosine), N,N-dimethylsphingosine, trimethyl sphingosine and analogs and derivatives ofsphingosine such as dihydrosphingosine, and myriocin. Fumonisins andfumonisin analogs, although they inhibit ceramide synthase, alsoincrease levels of sphinganine due to the inhibition of de novosphingolipid biosynthesis, resulting in apoptosis. Other molecules thatincrease ceramide levels include, without limitation, miltefosine(hexadecylphosphocholine). Sphingolipid modulators, such as thosementioned above, can be used in combination with one or more othersphingolipid modulators and/or with one or more chemotherapy agents of adifferent class(es).

In some embodiments, oligonucleotides are provided as cancer therapies.They include Genasense (oblimersen, G3139, from Genta), an antisenseoligonucleotide that targets bcl-2 and G4460 (LR3001, from Genta)another antisense oligonucleotide that targets c-myb. Other oligomersinclude, without limitation, siRNAs, decoys, RNAi oligonucleotides andthe like. Oligonucleotides, such as those mentioned above, can be usedin combination with one or more other oligonucleotide inhibitors and/orwith one or more chemotherapy agents of a different class(es).

Chemotherapy agents can include cocktails of two or more agents (e.g.,KBU2046 and a chemotherapeutic and/or hormone therapeutic). In severalembodiments, a chemotherapy agent is a cocktail that includes two ormore alkylating agents, platinums, anti-metabolites, anthracyclines,taxanes, camptothecins, nitrosoureas, EGFR inhibitors, antibiotics,HER2/neu inhibitors, angiogenesis inhibitors, kinase inhibitors,proteaosome inhibitors, immunotherapies, hormone therapies, photodynamictherapies, cancer vaccines, sphingolipid modulators, oligomers orcombinations thereof.

In several embodiments of the present invention, radiation therapy isadministered in addition to the administration of an oligonucleotidecompound. Radiation therapy includes both external and internalradiation therapies.

In some embodiments, of the present invention, surgery is used to removecancerous tissue from a patient. Cancerous tissue can be excised from apatient using any suitable surgical procedure including, for example,laparoscopy, scalpel, laser, scissors and the like. In severalembodiments, surgery is combined with chemotherapy. In otherembodiments, surgery is combined with radiation therapy. In still otherembodiments, surgery is combined with both chemotherapy and radiationtherapy.

Embodiments are described herein for inhibiting the movement of humanbreast, lung and colon cancer cells. Compositions and methods to such anend find use, for example, in: (1) inhibiting invasion of precancerouslesions (e.g., non-invasive lesions; also known as in situ lesions or insitu cancer), and inhibiting the formation of true, e.g., invasivecancer; (2) inhibiting organ confined cancer from invading outside ofthe local organ and invading into adjacent organs (e.g., inhibiting lungcancer from invading into the center of the chest where it can penetratethe aorta and cause death through resultant blood loss); and (3)inhibiting movement of cancer cells throughout the body (e.g., inhibitmetastasis). Compositions and methods find use in, for example, colon,breast and lung cancers, all of which commonly invade local organs,thereby inducing morbidity and mortality, and all of which metastasizethroughout the body, inducing morbidity and mortality. Experimentsconducted during development of embodiments described herein demonstrateinhibition of the function of heat shock protein 90 (HSP90) byinhibiting phosphorylation of its serine 226. The function of HSP90 isinhibited by increasing its ability to bind CDC37. A series of compoundshave been designed to increase HSP90/CDC37 interaction, and experimentshave demonstrated that they inhibit cancer cell motility. It has beendemonstrated that therapeutically inhibiting cancer cell motility can becombined with cytotoxic chemotherapy and that it increases iteffectiveness. It was been demonstrated that therapeutically inhibitingcancer cell motility can be combined with hormone therapy for prostatecancer, and that it increases it effectiveness. Further, the approachesdescribed herein can be applied to overcome resistance to hormonetherapy, and in a wide variety of cancers.

EXPERIMENTAL Example 1 Materials and Methods

Selection and Synthesis of Optimized Small Chemical Probes

Functional screens consisted of a Boyden chamber cell invasion assay(Craft et al., 2007; herein incorporated by reference in its entirety)for efficacy, and a three day cell growth inhibition assay (Liu et al.,2002; herein incorporated by reference in its entirety) for toxicity.Additional functional measures of toxicity included a NCI-based screenof the NCI 60-cell line panel (Shoemaker, 2006; herein incorporated byreference in its entirety), expression of estrogen-responsive genes byqRT/PCR (Ding et al., 2007; herein incorporated by reference in itsentirety), induction of an estrogen-responsive promoter by luciferaseassay (Breen et al., 2013; Catherino and Jordan, 1995; hereinincorporated by reference in their entireties), and a hematopoietic stemcell 14-day colony formation assay (Bergan et al., 1996; hereinincorporated by reference in its entirety). Protein Data Base (PDB)X-ray crystallographic structural data (PDB IDs: IX7R, IX7J) was used todetermine what chemical groups of genistein bound to the ER. Themigration of individual cells was assessed by time-lapse videomicroscopy.

Evaluation of KBU2046 Efficacy and Pharmacokinetics in Murine Models

Orthotopic implantation of human PCa cells and quantification of distantmetastasis was performed as described (Pavese et al., 2013; hereinincorporated by reference in its entirety). Orthotopic implantation ofhuman breast cancer cells, followed by surgical removal of resultantprimary tumor, was performed as described (du Manoir et al., 2006;herein incorporated by reference in its entirety). Quantification ofKBU2046 plasma concentrations was performed by LCMS. Resultantpharmacokinetic parameters were calculated with a three-compartmentmodel using a naive pooled data approach (Kataria et al., 1994; hereinincorporated by reference in its entirety), oral bioavailability wascalculated as described in Avram et al., 2009 (herein incorporated byreference in its entirety), model fitting used the extendedleast-squares maximum likelihood function with data weighted with theinverse of the model-based variance of the data at the observation times(Barrett et al., 1998; herein incorporated by reference in itsentirety), and model misspecification sought by inspection of themeasured and predicted findings (Cobelli and Foster, 1998; Foster, 1998;herein incorporated by reference in their entireties). Critical organswere examined for structural damage using a semi-quantitativehistological scoring system (Knodell et al., 1981; herein incorporatedby reference in its entirety), while their function was assessed bycomprehensive clinical laboratory testing of blood samples. All animalexperiments were conducted under protocols approved by the InstitutionalAnimal Care and Use Committee of Northwestern University.

Evaluating KBU2046's Ability to Inhibit the MKK4 Pathway

The ability of small chemicals to bind MKK4 was assessed by Fluorescencethermal shift assay (Krishna et al., 2013; herein incorporated byreference in its entirety) and by isothermal titration calorimetry(Polier et al., 2013; Zubriene et al., 2010; herein incorporated byreference in their entireties), their ability to inhibit MKK4 kinaseactivity was assessed by in vitro kinase assay (Krishna et al., 2013;herein incorporated by reference in its entirety), and their ability toinhibit cell signaling in cells was assessed by phosphoprotein Westernblot (Huang et al., 2005; Xu and Bergan, 2006; herein incorporated byreference in their entireties).

Using Proteomics to Identify the Pharmacologic Site of Action of KBU2046

Screening for changes in the kinome induced by KBU2046 were evaluatedwith the Kinoview™ assay system. Proteins pulled down by motif-directedKinoview™ antibody were identified through PhosphoScan™ technology,using LTQ-Orbitrap LC-MS/MS analysis coupled to a SEQUEST/Sorcerer dataanalysis suite (Lundgren et al., 2009; herein incorporated by referencein its entirety).

Defining the Protein Cleft where KBU2046 Binds

Stabilization of HSP90β/CDC37 heterocomplexes was evaluated by a drugaffinity responsive target stability (DARTS) assay (Lomenick et al.,2009; herein incorporated by reference in its entirety). Construction ofan in silico model used experimental data resulting from KBU2046compound structure, DARTS assays, crystal structures of human HSP90β(pDBs luym, 3nmq and 3pry) and of HSP82-CDC37 complex from yeast (PDBlus7), which were determined by X-ray diffraction-based crystallographicanalysis, and experimental probing of HSP90β structure using variedlength chemical cross-linkers (Chavez et al., 2013; herein incorporatedby reference in its entirety) coupled to MS3 analysis using ReACT(Weisbrod et al., 2013; herein incorporated by reference in itsentirety) in a manner that satisfies expected Protein InteractionReporter mass relationships (Tang et al., 2005; herein incorporated byreference in its entirety). Experimentally determined structuralinformation was then integrated and analyzed on the APPLIED Pipeline(Analysis Pipeline for Protein Ligand Interactions and ExperimentalDetermination) at the Argonne Leadership Computing Facility, ArgonneNational Laboratory. Building upon experimental findings, the analysisfollowed a multi-stage algorithm that took into considerationprotein-protein and protein-ligand interactions by combiningevolutionary protein surface analysis (Binkowski et al., 2003; Binkowskiand Joachimiak, 2008; Binkowski et al., 2005; herein incorporated byreference in their entireties), robust homology modeling (Leaver-Fay etal., 2011; herein incorporated by reference in its entirety), massivelyparallel docking simulations using mixed strategies (Deng and Roux,2008; Graves et al., 2008; Lang et al., 2009; Moni.s et al., 2009; Wanget al., 2006; herein incorporated by reference in their entireties), andadvanced, physics-based rescoring methodologies (Jiang et al., 2009;Jiang and Roux, 2010; Wang et al., 2006; herein incorporated byreference in their entireties).

Example 2 Chemical Synthesis

Exemplary Procedure for Large-Scale Production of 4′-Fluoroisoflavanone(KBU2046).

3-(dimethylamino)-1-(2-hydroxyphenyl)prop-2-en-1-one. The startingmaterials 2′-hydroxyacetophenone (50 mmol, 6.02 mL) andN,Ndimethylformamide dimethyl acetal (50 mmol, 6.64 mL) were added to a10-20 mL microwave vial. The vial was capped and heated in a BiotageInitiator microwave synthesizer at 150° C. and 11 bar for 10 minutes.The resulting dark orange liquid was allowed to cool to 23° C., at whichtime yellow-orange crystals crashed out of solution. The crystals werecollected and washed with hexanes (50 mL), then dried and weighed togive 3-(dimethylamino)-I-(2-hydroxyphenyl)prop-2-en-I-one (9.09 g, 95%)as orange-yellow needles. Product was confirmed by NMR andultra-performance liquid chromatography/mass spectrometry (UPLCMS).

3-bromochromone. 3-bromochromone was prepared by a procedure taken fromGammill (Gammill, 1979; herein incorporated by reference in itsentirety). To a flame-dried 250 mL round bottom flask, was added3-(dimethylamino)-1-(2-hydroxyphenyl)prop-2-en-1-one (36.6 mmol, 7.0 g),which was dissolved in CHCl₃ (70 mL). The reaction flask was cooled to0° C. in an ice bath, then Br₂ (36.6 mmol, 1.87 mL) was added dropwisethrough an addition funnel. After all of the Br₂ was added, water (70mL) was added slowly to the reaction and it was stirred at 23° C. for 10minutes. The dark orange/yellow organic layer was then separated fromthe aqueous layer, which was back extracted with 3×50 mL CHCl₃. Thecombined organic layers were then dried over Na₂SO₄ and concentrated togive a dark orange oil. This was purified by flash column chromatography(SiO₂ 10% EtOAc/hexanes) to afford 3-bromochromone (5.26 g, 64%) as anoff-white solid. Product was confirmed by NMR and ultra-performanceliquid chromatography/mass spectrometry (UPLCMS).Palladium Tetrakis(Triphenylphosphine) (Pd(PPh₃)₄).

The catalyst for the Suzuki-Miyaura cross-coupling reaction tosynthesize 4′-fluoroisoflavone was made using a procedure by Coulson(Coulson et al., 1990; herein incorporated by reference in itsentirety). To a flame-dried 100 mL Schlenk flask was added PdCl₂ (5mmol, 890 mg) and triphenylphosphine (25 mmol, 6.56 g). The solids weredissolved in DMSO (60 mL), then the mixture was purged with N₂ andheated to 145° C., at which time it turned a bright yellow-orange color.The reaction was removed from heat and allowed to stir at roomtemperature for 15 minutes, then hydrazine hydrate (20 mmol, 0.972 mL)was added via syringe, with a vent needle in place to account for theformation of N₂ gas. After the hydrazine hydrate had been added, thereaction was cooled to 23° C., during which time a yellow solid crashedout of solution. The solid was washed under Schlenk filtrationconditions with 2×50 mL EtOH, then 2×50 mL ether to yield Pd(PPh₃)₄(5.31 g, 94%) as a canary yellow solid that was stored under N₂.

4′-fluoroisoflavone. 4′-fluoroisoflavone was prepared on large scaleaccording to a procedure from Suzuki and Miyaura (Hoshino et al., 1988;herein incorporated by reference in its entirety). To a flame-dried 500mL round bottom flask was added 3-bromochromone (50 mmol, 11.25 g),4-fluorophenylboronic acid (55 mmol, 7.69 g) and NazC03 (100 mmol, 10.6g). The solids were dissolved in a mixture of benzene (100 mL) and water(50 mL), and the system was purged with N₂ for 10-15 minutes. ThePd(PPh₃)₄ catalyst (2.5 mmol, 2.89 g) was then added, at which time thereaction turned a bright orange. The flask was equipped with a refluxcondenser and the reaction was heated to reflux (80° C.) overnight.After approximately 16 h, the reaction was cooled to 23° C. and wasdiluted with EtOAc (250 mL), then the crude material was passed througha plug of silica with EtOAc as the eluent. The organic material wasdried over Na₂SO₄ and concentrated to give a dark brown solid that wasadsorbed onto silica gel using DCM. Material purified by flash columnchromatography (SiO₂, 20% EtOAc/hexanes) to afford 4′-fluoroisoflavone(8.14 g, 67% yield) as a yellow-orange solid that showed minorimpurities by ¹H NMR spectroscopy. Slightly impure material was takenonto the next step of the synthesis without further purification.Product was confirmed by NMR and ultra-performance liquidchromatography/mass spectrometry (UPLCMS).

4′-fluoroisoflavanone (KBU2046). To a flame-dried 500 mL round bottomflask was added 4′-fluoroisoflavone (25 mmol, 6.01 g), and the solid wasdissolved in dry THF (100 mL). The solution was cooled to −78° C. (dryice/acetone bath), monitored by a thermocouple. Once the solution hadcooled to the desired temperature, L-selectride (55 mmol, 55 mL, 1 Msolution in THF) was added dropwise over a period of 30-45 minutes. Thereaction was then allowed to stir at −78° C. for 2 h, after which timeit was quenched with MeOH (55 mL) at −78° C. The mixture was then pouredinto 300 mL of water, and the aqueous layer was adjusted to pH 7 with 2M HCl. The aqueous layer was extracted 2×200 mL with EtOAc, then thecombined organic layers were dried over Na₂SO₄ and concentrated to givea dark brown oily solid. This was purified by flash columnchromatography (SiO₂, 1:1 hexanes:DCM) to give 4.5 g of crude materialthat was recrystallized in hexanes to afford 4′-fluoroisoflavanone (3.4g, 56%) as a fluffy white solid. It was checked for purity by both ¹HNMR and HPLC analysis, with material that was >98% pure taken ontoanimal studies.

Related analog compounds were synthesized in addition to the parent4′-fluoroisoflavanone (KBU2046). These compounds were prepared in thesame general manner of KBU2046. The structure and purity of theadditional analogs were confirmed by NMR spectroscopy (¹H and ¹³C) aswell as by UPLCMS (minimal ion fragmentation). All compounds wereisolated and stored in powdered form (in the absence of light) and wereformulated into DSMO stock solutions just prior to use.

Example 3 KBU2046 Inhibition of Cell Motility and Metastasis

Identification of a Selective Inhibitor of Cell Motility and Metastasis

Flavonoids were selected as a platform to advance the synthesis ofprobes, for at least the reasons that they exert a wide range ofbiological effects, and because changes in their structure by a singleatom can significantly impact their spectrum of biological activity(Andersen and Markham, 2006; herein incorporated by reference in itsentirety). Consequently, they constitute biological probes withatom-level resolving capacity, and their chemical scaffold supports ahigh level of tailored, selective, medicinal chemistry refinement.4′,5,7-trihydroxyisoflavone (genistein) was selected as a startingpoint. It has been demonstrated that genistein inhibits human prostatecancer (PCa) cell invasion in vitro (Huang et al., 2005; hereinincorporated by reference in its entirety), and in the context of aprospective human trial that it down regulates matrix metalloproteinase2 (MJ\P-2) expression in prostate tissue (Xu et al., 2009; hereinincorporated by reference in its entirety). However, its known widespectrum of biological effects render it unusable as a selective andpotent biological probe (Pavese et al., 2010; herein incorporated byreference in its entirety). But, these very same properties optimize itspotential to selectively probe a wide spectrum of bioactive sites uponchemical diversification.

From genistein, chemical probes were developed by systematic medicinalchemistry diversification with iterative selection for inhibition ofhuman PCa cell invasion (FIG. 1). Along with this selectivity profiling,a major goal was deselection for inhibition of cell growth (an indicatorof off-target effects). Importantly, genistein is known to exertestrogenic effects (Messina et al., 2006; herein incorporated byreference in its entirety), and they were considered off-target withrespect to the goal of selectively modulating cell motility. Guided bythe crystal structure of the estrogen receptor (ER) with bound genistein(Manas et al., 2004; herein incorporated by reference in its entirety),ER-binding was deselected for. Using this strategy,(±)-3(4-fluorophenyl)chroman-4-one (KBU2046), a halogen-substitutedisoflavanone, was discovered (FIG. 1).

KBU2046 inhibits cell invasion equal-to-or-greater-than that ofgenistein for human prostate cells, including normal prostate epithelialcells, as well as primary and metastatic PCa cells (FIG. 2A). Cellmigration is a major determinant of cell invasion (Friedl and Wolf,2003; herein incorporated by reference in its entirety), and KBU2046inhibited the migration of human prostate, breast, colon and lung cancercells (FIG. 2B). Importantly, KBU2046 had high selectivity in cellularassays. It was not toxic to human prostate cells (FIG. 2C), to humanbone marrow stem cells (FIG. 2D), nor to cells in the NCI60 cell linepanel (FIG. 9). Toxicity to bone marrow is induced by a wide array ofdifferent therapeutic agents, and is a frequent and dose-limitingtoxicity of anti-cancer agents (Guest and Uetrecht, 1999; hereinincorporated by reference in its entirety). Furthermore, inestrogen-responsive human breast cancer MCF-7 cells, KBU2046 did notactivate estrogen-responsive genes (FIG. 2E; FIG. 9).

KBU2046 Selectively Inhibits Metastasis and Prolongs Survival

Probes were designed to contain chemical properties known to beassociated with systemically active small molecules (FIG. 10) (Lipinskiet al., 2001; herein incorporated by reference in its entirety).Employing an established orthotopic implantation murine model of humanPCa metastasis (Lakshman et al., 2008; herein incorporated by referencein its entirety), the ability of KBU2046 to inhibit the formation ofdistant metastasis was quantified. KBU2046 significantly decreasedmetastasis in a dose-dependent manner by up to 92%, at plasmaconcentrations of 1.1-24 nM (FIGS. 3A, 3B). Comprehensivecharacterization of KBU2046 pharmacokinetics in mice demonstratedmaintenance of plasma concentrations >24 nM for 9.3 hours after a singleoral dose, and allowed characterization of pharmacokinetic parameters(FIG. 3C; FIG. 11). At the systemic level, KBU2046 was a highlyselective inhibitor of metastasis. Comprehensive analysis of primarytumor growth, animal behavior, weight, histologic examination ofmultiple organs and serum chemistry profiling, failed to identifyKBU2046-associated of f-target effects (FIG. 12, FIG. 13).

Recognizing the established link between metastasis and decreasedsurvival in humans, the impact of KBU2046 on survival was assessed. Theorthotopic PCa model exhibits rapid tumor growth around the urogenitaltract, precluding assessment of the impact of metastatic burden onsurvival. However, orthotopic implantation of human breast cancer cells,followed by surgical removal of the resultant primary tumor, provides amurine model wherein survival is dictated by metastatic burden (duManoir et al, 2006; herein incorporated by reference in its entirety).KBU2046 significantly prolonged the survival of mice treated in apost-surgery adjuvant setting (FIG. 3D).

KBU2046 Inhibits Invasion by Decreasing Phosphorylation of Ser226 onHSP90β

Low nanomolar concentrations of genistein inhibited the kinase activityof mitogen-activated protein kinase kinase 4 CMXK.41MAP2K4/MEK4) (Xu etal., 2009; herein incorporated by reference in its entirety), in turninhibiting downstream phosphorylation of p38 MAPK (Huang et al., 2005;herein incorporated by reference in its entirety) and of heat shockprotein 27 (HSP27) (Xu and Bergan, 2006; herein incorporated byreference in its entirety), which in turn inhibits MMP-2 expression andcell invasion in vitro, with systemic effects translating intoinhibition of human PCa metastasis in mice (Lakshman et al., 2008;herein incorporated by reference in its entirety) and inhibition ofMMP-2 expression in human prostate tissue (Xu et al., 2009; hereinincorporated by reference in its entirety). In contrast to genistein,KBU2046 did not bind to MKK4 or inhibit its kinase activity in vitro,and it did not inhibit phosphorylation of p38 MAPK or of HSP27 in cells(FIG. 4). This finding, while surprising, demonstrates that the probestrategy de-selected for inhibition of the MKK4 signaling axis. Thisprovides a measure of the unbiased nature of the small chemical probestrategy.

Because KBU2046 does not target MKK4, alternative methods foridentifying its biological target(s) were pursued. The KinomeView® panelof antibodies (Cell Signaling Technology, Inc.) detects establishedprotein phosphorylation motifs, and were was to probe forKBU2046-induced changes in protein phosphorylation (FIG. 5A; FIGS.14A-D). Phosphoprotein changes that met the following criteria wereprioritized: (1) changes were observed in cells and in tumors of treatedmice (from FIG. 3A), (2) changes counteracted transforming growth factorβ (TGFβ)-induced effects, and that were reproducible. TGFβ is ubiquitousin vivo, is known to increase PCa cell invasion (Huang et al., 2005;herein incorporated by reference in its entirety), and KBU2046'santi-invasion efficacy is greater in the presence of TGFβ (FIG. 15).Genistein was evaluated under identical treatment conditions forcomparison. Its many pharmacologic effects induced widespread changes inprotein phosphorylation (FIG. 14A-D). In contrast, with KBU2046, only adecrease in intensity of an 83 kDa protein band met the pre-specifiedcriteria (arrow in FIG. 5A). The high molecular selectivity of KBU2046was further supported by its failure to inhibit over 400 differentprotein kinases and 20 phosphatases examined, in three different invitro assay systems.

The 83 kDa protein was identified by pretreating PC3 cells with KBU2046or vehicle control, treating with TGFB and performing LC-MSIMS analysison proteins pulled down by the KinomeView® antibody used in FIG. 5A.Resultant data were analyzed with a SEQUEST/Sorcerer data analysis suite(Lundgren et al., 2009; herein incorporated by reference in itsentirety), and proteins further selected based upon predeterminedparameters, including expression at >1=3× background levels, exhibitinga >1=2.5 fold decrease with KBU2046 and within ±5 kDa of the 83 kDaindex band. This approach yielded a single protein, HSP90β, andindicated that KBU2046 decreased the abundance of phosphorylated Ser226on HSP90β by 6.6 fold (FIG. 5B; FIG. 16).

Using a (S226A)-HSP90β construct, we demonstrated that loss of Ser226inhibited cell invasion compared to wild type (WT)-HSP90 β, and that itsloss abrogated KBU2046 efficacy (FIG. 5C). The selectivity of HSP90B inmediating KBU2046 efficacy was further supported by demonstrating thatsiRNA-mediated HSP90 β knockdown inhibited cell invasion and abrogatedKBU2046 efficacy (FIG. 12). Importantly, HSP90 β-specific siRNA did notknockdown HSP90α. We next demonstrated that cells transfected with(S226D)-HSP90β, which provides a biological mimic of phosphorylatedSer226, exhibited increased invasion and were not sensitive to theeffects of KBU2046 (FIG. 5D). These findings identify phosphorylation ofHSP90β Ser226 as a regulator of cell invasion, and demonstrate that itis necessary and sufficient for mediating KBU2046 efficacy.

KBU2046 Binds to the CDC37/HSP90β Heterocomplex

With phosphorylation of HSP90β Ser²²⁶ identified as the regulator ofKBU2046 action, we sought to gain further insight into the interactionbetween KBU2046 and its protein target(s). Along with HSP90β, weconsidered CDC37 as a possible target. CDC37 is a co-chaperone thatmediates the binding of over 350 client proteins to HSP90β, includingover 190 kinases (Taipale et al., 2012; herein incorporated by referencein its entirety). The flexible arm-like structure of CDC37 (protein databank (PDB) ID: 2WOG) enables dynamic binding of large numbers ofkinases, and defines their positioning relative to Ser²²⁶. Anothermeasure of CDC37's mobile nature is that its conformational changes arecoupled to that of the highly dynamic HSP90 chaperone cycle (Vaughan etal., 2006; herein incorporated by reference in its entirety). Thecoordinated and dynamic movements of CDC37 and HSP90β dictate thespectrum of kinases that are juxtaposed to, and able to modulate, Ser²²⁶phosphorylation status. It was reasoned that if KBU2046 bound to eitherCDC37 or HSP90β, the new chemical interactions that resulted could alterthe dynamics and functions of these two proteins, thus regulating kinaseaccessibility to Ser²²⁶ and its phosphorylation.

There was no evidence of KBU2046 binding to either CDC37 or HSP90β byisothennal titration calorimetry, by fluorescence-based thermal shiftassay, or by drug affinity responsive target stability (DARTS) assay(FIG. 18) (Lomenick et al., 2009; herein incorporated by reference inits entirety). DARTS provides a sensitive measure of ligand-inducedchanges in protein structure and dynamics by measuring the ability of aligand to protect its target from protease digestion (Lomenick et al.,2009; herein incorporated by reference in its entirety). AlthoughKBU2046 did not bind CDC37 or HSP90β individually, CDC37 and HSP90βassociate to form a tetrameric hetero-complex (Vaughan et al., 2006;herein incorporated by reference in its entirety). In a DARTS assaycombining CDC37 and HSP90β, KBU2046 protected both proteins fromdigestion (FIG. 6A). The intensity of CDC37 increased, that of theHSP90β degradation product decreased, and both effects werestatistically significant, concentration-dependent and were evident at100M. The high selectivity of KBU2046 for protein binding was furthersupported by synthesizing a biotin chemical linker to KBU2046,demonstrating that it retained biological activity, demonstrating thatit bound to intact cells (e.g., under physiological conditions ofCDC37/HSP90β heterocomplex formation), and demonstrating that it failedto bind to an array of over 9,000 human proteins (FIG. 19A-D).

Together, these findings demonstrate that KBU2046 is operating in adistinct fashion from that of classical HSP90 inhibitors (Neckers andWorkman, 2012; Whitesell et al., 2012; herein incorporated by referencein its entirety). Instead of binding directly to HSP90α and inhibitingthe chaperone cycle, which in turn induces cytotoxicity, KBU2046inhibits phosphorylation of HSP90βSer²²⁶. This residue is not present onHSP90α. Further, KBU2046 does not bind to isolated HSP90β, nor does itinduce cytotoxicity. Finally, and importantly, KBU2046 binds to andstabilizes the CDC37/HSP90β heterocomplex.

These combined experiments indicate that KBU2046 is binding in a cleftthat is only present when CDC37 and HSP90β interact. A comprehensiveanalysis of HSP90β and CDC37 experimental structural information,including X-ray crystallographic data (PDB IDs: luym, 3nmq, 3pry, 2cg9and lus7) (Roe et al., 2004; Vaughan et al., 2006; Wright et al., 2004;herein incorporated by reference in their entireties) and chemicalcross-linker physical mapping analysis (Chavez et al., 2013), indicatesthat CDC37/HSP90β heterocomplex formation results in the formation of anew pocket that modeling studies indicate allows for KBU2046 bindingwithout any high energy steric interactions, and with a favorable energyscore (FIGS. 6B, 6C, 6D; FIG. 20). In this computational arrangement,Arg¹⁶⁷ from CDC37 protrudes into a large cleft, engages in a hydrogenbond with the carboxyl side chain of Glu³³ from HSP90β, which promotesformation of a new pocket, into which KBU2046 binds.

KBU2046 does not Inhibit Kinase or Phosphatase Activity.

Three different assays systems were employed to detect inhibition.

Kinase Assay System #1.

The KINOMEscan™ assay (Ambit Biosciences). This assay evaluates 442kinases, including 400 distinct parental kinases plus mutants known toalter activity or responsiveness. It does so in the context of an assaythat measures the ability of putative inhibitors to inhibit binding ofbacterial purified kinase to immobilized phospho-acceptor proteinsubstrate. This approach has been successfully used by multiple groupsto identify kinase interactions of several small molecule kinaseinhibitors (Fabian et al., 2005; Karaman et al., 2008; hereinincorporated by reference in their entireties). KBU2046 was tested at 10μM. This assay was completely negative. There were two initial falsepositives (for a false positive rate of 0.4%), that were subsequentlyfound to be negative in a dedicated follow up analysis. Specific andimportant negative findings include MKK4 and p38 MAPK (α, β, and γisoforms). There was no evidence that KBU2046 inhibits kinase functionby competing for phospho-acceptor binding.

Kinase Assay System #2.

The Kinex™ kinase assay system (Kinexus Proteomics Company). Thisplatform uses recombinant human protein kinases expressed in an insectexpression system, thus allowing an avenue for post-translationalmodification. Also, this platform measures inhibition of ATP binding.This platform putatively measured 200 different kinases, and we testedKBU2046 at 1 and 10 μM. This assay was also completely negative. Thisscreen was informative for a number of kinases (e.g., where controlswere active and at KBU2046 concentrations that did not interfere withdetection). KBU2046 did not inhibit p38 MAPK (all isoforms) nor MKK4.There was no evidence that KBU2046 inhibits kinase function by competingfor ATP binding in the active site.

Kinase Assay System #3.

KinaseProfiler™ and PhosphataseProfiler™ assay platforms (Millipore).This platform is radiometric-based (considered gold standard). Itmeasures competition with respect to ATP for kinases and substrateprotein for phosphatases. Most proteins were expressed via aninsect-based system, and it evaluates a panel of 284 kinases and 20phosphatases. There were two initial false positives, for a falsepositive rate of 0.7%, but both failed to be confirmed upon in-depthinvestigation. Important negative findings include: MKK4, MKK6, p38MAPK, MAPKAPK2, ERK, MEK1, JNK1, 2 and 3, and numerous other MAPKcascade-associated kinases. There is no evidence that KBU2046 inhibitskinase activity by competing for ATP binding in the active site. Noevidence supports inhibition of phosphatase activity.

Construction of Structural Model of HSP90 β, CDC37 and KBU2046Interaction.

Analysis began with experimentally determined structural information,including the crystal structures of human HSP90 β (pDBs luym, 3nmq and3pry) and HSP82⋅CDC37 complex from yeast (pDB Ius7), which weredetermined by X-ray diffraction-based crystallographic analysis. TheHSP90β structure was probed using chemical cross-linking with massspectrometry, employing chemical cross-linkers of various lengths, aspreviously described (Chavez et al., 2013; herein incorporated byreference in its entirety). In addition, the HSP90β structure was probedin human cells with the use of chemical cross-linkers called ProteinInteraction Reporters (PIRs) (Chavez et al., 2013; herein incorporatedby reference in its entirety). Cross-linked peptide samples wereanalyzed using ReACT (Weisbrod et al., 2013; herein incorporated byreference in its entirety) which allows targeted MS3 to be carried outefficiently on each released peptide that satisfies expected PIR massrelationships (Tang et al., 2005; herein incorporated by reference inits entirety).

It was found that KBU2046 does not bind directly to HSP90β or CDC37(FIG. 18), but that it does bind to the HSP90β-CDC37 complex (FIG. 6A).Therefore, the complex affords a suitable binding pocket that is notindependently present on either protein. In the absence of a complete,X-ray crystallographic structural model of HSP90β, a homology basedmodel using existing structures from the protein data bank (PDB) wasrelied upon. Structures of the human HSP90β N-terminal ATPase domain(PDB ids=luym, 3nmq) and middle domain (PDB id=3pry) were used. Thenoncontiguous models cover 65% of the primary sequence, separated by ahighly disordered region of 63 residues that terminates the ATP bindingdomain. No experimental models exist for the C-terminal region. Theentirety of the HSP90β structure was then modeled against the HSP82template from S. cerevisiae (PDB id=2cg9) (Leaver-Fay et al., 2011). Amodel of the complex of HSP90 β-CDC37 was completed throughsuperposition of the HSP90β model onto the structure of the S.cerevisiae HSP82-CDC37 complex (PDB id=lus7). The sequence identitybetween HSP90β and HSP82 is 94% at the CDC37 interface (86% for entireprotein), thus preserving the integrity of the interactions. A markedfeature of the HSP90 structure is the nucleotide binding site. The site,with solvent accessible area of 496.2 Angstroms² and volume of 301.3Angstums³ (Binkowski et al., 2003b; herein incorporated by reference inits entirety), has been well characterized and targeted by a variety ofcompounds for anti-cancer activity. When complexed with CDC37, anexpansive surface, with solvent accessible area of 1446.4 Angstroms² andvolume of 2082.6 Angstroms³, is formed at the interface (FIG. 20B).Arg167_(cdc37) is drawn in to the nucleotide binding pocket and forms ahydrogen bond with the carboxyl side chain from Glu33_(HSP90) (Roe etal., 2004; herein incorporated by reference in its entirety).Arg167_(cdc37) does not preclude access to the nucleotide binding siteor displace any bound ligands (Roe et al., 2004; herein incorporated byreference in its entirety). It does, however, divide the large cleftinto two distinct pockets: a newly formed pocket and the undisturbed,yet smaller, nucleotide binding site. The new pocket has solventaccessible area of 429.2 Angstroms² and volume of 832.5 Angstroms³ andmeets the criteria of a structural feature only present in theHSP90β-CDC37 complexed state.

Example 4 Downstream Regulators

Having identified inhibition of Ser²²⁶ phosphorylation as the moleculartarget of KBU2046 for inhibition of cell motilty, experiments wereconducted during development of embodiments herein to identifydownstream regulators of its action. PC3 cells with KBU2046, andscreened for differentially expressed genes using two differentSABiosciences (Qiagen) gene array platforms: Human Metastasis Array andHuman Extracellular Matrix and Adhesion Molecule Array. KBU2046 wasfound to significantly suppress osteonectin expression by 2.0 fold. Noother significant effects were identified. Separate experimentsconfirmed differential expression by gene-specific qRT/PCR (FIG. 21A).Osteonectin is an extracellular matrix protein that when overexpressedin PCa has been shown to increase both cell motility and invasion.Clinically, overexpression of osteonectin in primary PCa has beenassociated with the development of metastasis to bone. Together, thesefindings indicate that KBU2046 antimetastatic efficacy is mediated, atleast in part, by suppression of osteonectin, which was confirmed bydemonstrating that siRNA-mediated suppression of osteonectin inhibitshuman PCa cell invasion and abrogates KBU2046 therapeutic efficacy(FIGS. 21 B and C).

KBU2046 Disrupts HSP90β Chaperone Function

Biochemical methods were employed in experiments conducted duringdevelopment of embodiments herein to demonstrate that KBU2046 isaltering HSP90β/CDC37 heterocomplex formation and resultant chaperonefunction, that it does so across model systems whose findings arecorroboratory, that it does so under rigorously defined in vitro systemsutilizing recombinant purified proteins, and that it does so in intactcellular systems. At least the latter involves clinically relevantscenarios related to PCa through a primary effect upon androgen receptor(AR) biology, relating to the fact that AR is a client protein whosefunction requires HSP90 chaperone activity. Further, findings indicatethat KBU2046 binds within a cleft that is formed between the interfaceof HSP90β and its co-chaperone, CDC37.

CDC37, a co-chaperone, mediates binding of over 350 client proteins toHSP90β, inclusive of over 190 kinases. Its arm-like structure (pdb ID:2WOG) enables highly dynamic and kinetic conformational changes relatedto the binding of large numbers of kinases, bringing them injuxtaposition to Ser²²⁶. It was contemplated that if KBU2046 bound toeither CDC37 or HSP90β, the resultant chemical bonds would serve toalter the dynamic function of these two proteins, and their regulationof kinase accessibility to Ser²²⁶. An exhaustive battery of assaysdesigned to detect KBU2046 binding to HSP90β, CDC37, or HSP90β/CDC37heterocomplexes was conducted, including: fluorescence-based thermalshift assay, dynamic light scattering, isothermal titration calorimetry,bio-layer interferometry. Assays based on physical measures of bindingwere negative. However, several biochemical measure did detect binding.

The drug affinity responsive target stability (DARTS) assay measures theability of a bound ligand to protect a target protein from proteasedigestion, and provides a sensitive measure of ligand-induced changes inprotein structure, being particularly sensitive to changes in proteinflexibility. Considering that CDC37 and HSP90β bind to form aheterocomplex, it was demonstrated that KBU2046 bound toheterocomplexes. When both proteins were present in a DARTS assay,KBU2046 protected both from protease digestion, significantly increasingCDC37 protein and decreasing HSP90β degradation product in aconcentration-dependent fashion (FIG. 22A). When CDC37 or HSP90β wereexamined individually (e.g., not in a heterocomplex), no protection wasobserved.

A chemical linker to was attached KBU2046 (FIG. 23), that retainedbiological activity and that bound to intact cells (e.g., withphysiologic CDC37/HSP90β heterocomplexes), and experiments indicatedthat it did not bind a 9,000 human protein array (ProtoArray®;Invitrogen), inclusive of HSP90β and CDC37.

Experiments conducted during development of embodiments hereindemonstrated that KBU2046 regulates heterocomplex function. In an invitro system of recombinant HSP90β, CDC37 and casein kinase (CK)proteins (engineered by us), KBU2046 increased CK-mediatedphosphorylation of HSP90β (FIG. 22B). CDC37 mediates binding of clientproteins to HSP90β, that CK is a client protein kinase. KBU2046 does notmodulate CK kinase activity. CK did not increase phosphorylation ofSer226. These findings all indicate that inside the cell KBU2046stabilizes CDC37/HSP90β heterocomplex formation, this increases bindingof certain client proteins, thereby inhibiting that of other clientproteins which phosphorylate Ser226.

HSP90β and CDC37 structural information derived by a crosslinkingapproach wherein chemical cross-linking probes (of various lengths andligand binding capacities) are coupled to MS3-based analysis, X-raycrystallographic structural information (Protein Database ID's: luym,3nmq, 3pry and lus7), and ligand binding information informed by theDARTS experiments above. The structural information was integrated,through the APPLIED Pipeline platform at the Argonne National LaboratoryLeadership Computing Facility. It integrates protein surface analysis,robust homology modeling, massively parallel docking simulations usingmixed strategies, and advanced physics-based rescoring methodologies.The resultant informed model indicated that HSP90β and CDC37 bindingforms a new pocket into which KBU2046 binds without high-energy stericinteractions and with a favorable energy score (FIG. 22C). Arg167 fromCDC37 protrudes into a large cleft, hydrogen bonds with Glu33 of HSP90β,and forms a new pocket. This model indicates that KBU2046 promotesprotein-protein interaction. This model agrees with experimental DARTSdata demonstrating that KBU2046 stabilizes HSP90β/CDC37 heterocomplexes(FIG. 22A), but does not stabilize HSP90β or CDC37 individually.

Having demonstrated that KBU2046 impacts chaperone function in cell freesystems, experiments were conducted during development of embodimentsherein using molecular and cellular-based assays to demonstrate theeffect in cells. The chaperone action of HSP90 maintains AR in itsactive conformation, and inhibitors of HSP90 function blockligand-independent AR signaling. Therefore, if KBU2046 disrupts HSP90function, as experiments conducted indicate, KBU2046 would disruptAR-associated chaperone function, inhibit AR-dependent transcriptionalactivity and inhibit AR-dependent cell growth and enhance thetherapeutic efficacy of AR-directed therapy; the experiments conductedduring development of embodiments herein demonstrate as much.

Classic HSP90-targeting agents directly bind HSP90, interrupt itschaperone cycle, and thereby exert relatively profound changes on acentral cellular process, which in turn induces direct cytotoxicity. Atthe systemic level, such profound pharmacologic effects disrupt normalcellular function, induce systemic toxicity, and thereby limit ultimatetherapeutic efficacy. This is similar to the situation with cytotoxicchemotherapy. In contrast, KBU2046 represents a therapeutic manipulationthat exerts a more selective effect upon HSP90 function and is notsystemically toxic. However, one consequence of KBU2046's mechanisticeffects is that its effects upon rapid-readout in vitro assay systemsare less robust than cytotoxic chemotherapy or classic HSP90 inhibitors.Based on this, the experiments described herein involvingKBU2046-mediated disruption of AR signaling were expected to procudesmall effects with short term in vitro assay, as was observed. However,corroboratory effects were demonstrated across multiple assays,involving measures of molecular interaction, cell signaling and cellgrowth. Further, even in the context of short term in vitro assaysystems, enhanced efficacy with increased treatment time was observed.

Specifically, performed FLAG IP followed by silver stain was performedusing cells transfected with FLAG-HSP90β and treated with +/−KBU2046.KBU2046 disrupted binding of a ˜60 kDa protein (FIG. 24A). MS-basedproteomic analysis identified it a HOP. This is a very importantfinding. HOP is a member of the chaperone complex that brings AR toHSP90. Findings were confirmed by HOP-specific Western blot FIG. 24B).Thus, KBU2046 disrupts a molecular interaction known to be necessary forchaperone-mediated maintenance of active AR. KBU2046 disruption ofAR-related chaperone activity decreases AR-responsive gene activation(FIG. 24C). AR positive LNCaP cells were grown under charcoal strippedserum (CSS) hormone-free conditions, treated with KBU2046, R1881 (e.g.,androgen) and/or bicalutamide for 1 or 3 days and the expression ofAR-responsive prostate specific antigen (PSA) measured by qRT/PCR.KBU2046 had no effect by itself, but it inhibited R1881-mediated PSAexpression, with effects greater at 3 days. Though a weak agonist byitself in LNCaP cells (due to an AR T868A mutation), bicalutamidefunctionally inhibits the strong agonist action of R1881, and thusdecreases R1881-mediated increases in PSA. Importantly, 3 days treatmentwith KBU2046 increases the therapeutic efficacy of bicalutamide. KBU2046acts to inhibit androgen-driven cell growth, that it enhancesbicalutamide efficacy, and that effects are amplified as treatment timeincreases from 3 to 6 days (FIG. 24D). Assays were performed this inboth LNCaP (mutant AR) and VCaP cells.

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The invention claimed is:
 1. A method for treating a subject sufferingfrom cancer comprising administering the subject a compound havingformula of:

wherein the cancer is not prostate cancer or breast cancer.
 2. Themethod of claim 1, wherein the compound is administered prior tosurgical removal of a tumor.
 3. The method of claim 1, wherein thecompound is administered after surgical removal of a tumor.
 4. Themethod of claim 1, wherein the compound is co-administered with a secondcancer therapeutic agent.
 5. The method of claim 4, wherein the secondcancer therapeutic agent is a chemotherapeutic agent.
 6. The method ofclaim 4, wherein the second cancer therapeutic agent is an anti-motilityagent.